Amino acid sequences directed against a metalloproteinase from the adam family and polypeptides comprising the same for the treatment of adam-related diseases and disorders

ABSTRACT

The present application relates to amino acid sequences that are directed against and/or that can specifically bind to metalloproteinases from the ADAM family, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such ammo acid sequences, and to methods of preparing the same.

The present invention relates to amino acid sequences that are directedagainst (as defined herein) metalloproteinases from the ADAM family, aswell as to compounds or constructs, and in particular proteins andpolypeptides, that comprise or essentially consist of one or more suchamino acid sequences (also referred to herein as “amino acid sequencesof the invention”, “compounds of the invention”, and “polypeptides ofthe invention”, respectively).

The invention also relates to nucleic acids encoding such amino acidsequences and polypeptides (also referred to herein as “nucleic acids ofthe invention” or “nucleotide sequences of the invention”); to methodsfor preparing such amino acid sequences and polypeptides; to host cellsexpressing or capable of expressing such amino acid sequences orpolypeptides; to compositions, and in particular to pharmaceuticalcompositions, that comprise such amino acid sequences, polypeptides,nucleic acids and/or host cells; and to uses of such amino acidsequences or polypeptides, nucleic acids, host cells and/orcompositions, in particular for prophylactic, therapeutic or diagnosticpurposes, such as the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

The ADAM metalloproteinases disintegrins form a well-known family ofproteases (or proteinases) that mediates ectodomain shedding. Many ofthe processing events that include the release of cytokines, shedding ofcell surface molecules, release of growth factors and cleavage ofamyloid precursor protein (APP), are all shown to be mediated bymetalloproteases. Reference is for example made to the reviews byHuovila et al., Trends Biochem Sci, (30), 7, 413-22 (2005); Seals andCourtneidge, Genes Dev 17, 7-30 (2003); Fox and Serrano, Toxicon 45,969-85 (2005); Moss et al., Drug Discov Today, (6), 8, 417-426 (2001);Moss et al., Biochemistry, (43), 23, 7227-35 (2004); Handsley et al. IntJ Cancer, (115), 6, 849-60 (2005); Herren, News Physiol Sci, (17), 73-6(2002); and Hojilla et al., Br J Cancer, (89), 10, 1817-21 (2003); aswell as to the further references cited therein and the further priorart cited in the present specification. Reference is for example made toFIG. 1 in the review by Huovila et al., supra; FIGS. 2 and 3 in Allinsonet al., J Neurosci Res 74, 342-52 (2003); to FIGS. 1 and 3 in the reviewby Moss et al (2004), supra; to FIGS. 1 and 3 from the review by Sealsand Courtneidge, supra; and to FIG. 1 in the review by Herren, supra.

The ADAM (A disintegrin And Metalloproteases) family includes proteinscontaining a disintegrin-like and metalloprotease-like domain). TheADAMTS contain a thrombospondin (TS) domain in addition to the othercommon domains (and should be considered included within the term ADAMproteinase as used in the specification and claims). Together with theSnake Venom Metalloproteinases (SVMPs), ADAM and ADAM-TS form thereprolysin subfamily of metalloprotease. All family members share acommon multi-domain structure minimally containing a Pro domain, ametalloprotease (-like) and a disintegrin-like domain. Some ADAM haveadditional domains like cystein-rich and a transmembrane and cytoplasmicdomains. Reference is again made to FIG. 1 in the review by Huovila etal., supra; FIGS. 2 and 3 in Allinson et al., J Neurosci Res 74, 342-52(2003); to FIGS. 1 and 3 in the review by Moss et al (2004), supra; toFIGS. 1 and 3 from the review by Seals and Courtneidge, supra; and toFIG. 1 in the review by Herren, supra.

The substrates of ADAM metalloproteinases are also well-known, and forexample include CD23, TNFR1, IL1-RII, CX3CL1, TNF-alpha, TNFR1, TNFRII,and TRANCE, as well as the substrates mentioned in Table 1 of the reviewby Huovila et al., supra.; and Table 1 in the review by Moss et al(2004), supra.

Members of the ADAM family are also well-known in the art, and forexample and without limitation include:

-   -   ADAM1, ADAM2, ADAM3B, ADAM4, ADAM5, ADAM6, ADAM7, ADAM8, ADAM9,        ADAM10, ADAM11, ADAM12, ADAM13, ADAM14, ADAM15, ADAM16, ADAM17,        ADAM18, ADAM19, ADAM20, ADAM21, ADAM21, ADAM22, ADAM23, ADAM24,        ADAM25, ADAM26, ADAM27, ADAM28, ADAM29, ADAM30, ADAM31, ADAM32,        ADAM33, ADAM34, ADAM35, ADAM36, ADAM37, ADAM38, ADAM39, ADAM40    -   ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7,        ADAMTS8, ADAMTS9, ADAMTS10, ADAMTS11, ADAMTS12, ADAMTS 13,        ADAMTS 14, ADAMTS15, ADAMTS 16, ADAMTS 17, ADAMTS 18, ADAMTS 19,        ADAMTS20

Reference is also made to the following websites:http://merops.sanger.ac.uk/ andhttp://www.uta.fi/%7Eloiika/ADAMs/HADAMs.htm.

Diseases and disorders in which ADAM metalloproteinases are involved asalso well-known in the art. Reference is again made to the reviewsmentioned above and the further references cited therein; and also tofor example to Levy et al., Nature 413, 488-94 (2001); Stanton et al.;Nature 434, 648-52 (2005); Glasson et al., Nature 434, 644-8 (2005),Weskamp et al., Nat Immunol 7, 1293-1298 (2006); Allinson et al., JNeurosci Res 74, 342-52 (2003); and Blobel, Nat Rev Mol Cell Biol 6,32-43 (2005); and for example include:

-   -   ADAM8: allergy, inflammation (asthma, TRAPS)    -   ADAM9: hematological malignancies    -   ADAM10: chemotaxis, inflammation (asthma, rhinitis) Alzheimer    -   ADAM17/TACE: inflammation, IL1beta signaling, sepsis,        inflammatory, arthritis, diabetes, HIV cachexia, cancer and        TNF/EGF dependent diseases    -   ADAM-TS5: aggrecanase 2 and osteoarthritis, arthritis    -   ADAM15: atherosclerosis    -   ADAM33: Asthma

For example, ADAM17 is the major physiological TNFalpha convertingenzyme and is therefore essential to TNFalpha signaling. Therefore,inhibition of ADAM17 will be useful in any disease state where TNFantagonist has been validated, such as arthritis, diabetes, HIVcachexia, sepsis and cancer. Reference is for example made to the reviewarticles cited above and the further references cited herein and in thepresent specification. Moss et al. (2001) and Moss et al. (2004).

Also ADAM17 is responsible for the processing and release of TGFalphaand EGF. Indeed ADAM17−/− mice are embryonic lethal with phenotypereminiscent of the TGFalpha−/− and EGFR−/− mice. “Key functions of ADAMshave emerged in ErbB signalling pathways as being sheddases for multipleErbB ligands. As the ErbB pathway is a validated target for anti-cancerdrugs, the upstream activators of ErbB ligands, their sheddases, nowenter the spotlight as new drug targets in the ErbB pathway. ADAMs areinvolved not only in tumour cell proliferation but also in angiogenesisand metastasis. Therefore, strategies targeting ADAMs might be animportant complement to existing anti-ErbB approaches.” (See Blobel,supra).

ADAM10 and ADAM17 have been described as important sheddases forcytokines and their receptors, suggesting that these ADAMs are keymodulator of cytokines in vivo.

The gene ADAM33, was identified as an asthma susceptibility geneinvolved in airway remodeling.

Increasing evidence point that ADAM10 (and to lesser extend ADAM17) isan alpha-secretase of the amyloid precursor protein (APP). Becausecleavage of APP at the alpha site is believed to preclude cleavage atthe beta and gamma site (cleavage sites promoting the APP aggregationleading to Alzheimer disease), ADAM10 can be considered as a protectivefactor in the etiology of Alzheimer's disease. Activation of ADAM10might prove useful for the treatment of Alzheimer's disease (see forreview Allinson 2003 and ref therein).

ADAM12 cleaves and inactivate IGFBP3, a natural inhibitor of IGF1 and 2.Because low level of IGF are associated with osteoarthritis anddiabetes, the balance between IGFs and IGFBPs is crucial. Interestingly,mice overexpressing ADAM12 develop more abdominal and body fat. AlsoADAM12−/− mice have reduced adipocytes and partial defect in myogenesis.

ADAM9 is expressed in hematological malignancies and might process theEGFR ligand HB-EGF.

ADAM8 is a serological and histochemical marker for lung cancer(Ishikawa N, 2004). ADAM8 may be the CD23 shedding enzyme. Soluble CD23released from cells during allergic response stimulate IgE production.Specific inhibition of CD23 sheddase activity would be an importanttreatment of allergic reactions.

ADAM8 is (over)expressed in tissues around aseptically loosened totalhip implants, which are characterized by chronic foreign bodyinflammation and peri-implant bone loss. This is compatible with a rolefor ADAM8 in the formation of foreign body giant cells and osteoclasts.

Higher ADAM19 expression has been associated with clinical andstructural deterioration involved in renal disease, and ADAM19 may havea role in the dysfunctional renal allograft state.

In addition, expression levels and activities ADAM8 and ADAM19 areassociated with invasiveness in human primary brain tumors.

Snake venom metalloproteinases play an important role in viperidenvenoming (hemorrhage, edema, hypertension, hypovolemia, inflammationand necrosis) (see for example Fox et al., supra).

In mice, ADAM-TS5 is the major aggrecanase (aggrecan is a majorcomponent of the cartilage extracellular matrix) responsible ofosteoarthritis and inflammatory arthritis.

ADAMTS-13 deficiency gives rise to thrombotic thrombocytopenic purpura(TTP), a life threatening condition (see for example Levy et al., supra)

Also, of course, modulation of ADAMs may have an influence on thebiological pathways, physiological effects, signaling mechanisms andother biological and physiological activities that their respectivesubstrates (and the ligands of such substrates) are involved in. Thus,it is expected that modulation of ADAMs could also play a part in theprevention and treatment of diseases and disorders in which theirsubstrates are involved.

Another class of metalloproteinases closely related to the ADAM familyare the snake venom metalloproteinases such as, without limitation,Aclpref, Acostatin, Acurhagin, Acutolysin A, Atrolysin, Atrolysin A,Atrolysin B, Atrolysin C, Atrolysin E, Atroxase, BAP1, Berythractivase,Bilitoxin-I, Bothropasin, Brevilysin H6, Catrocollastatin,Contortrostatin, Ecarin, Fibrolase, Flavoridin, Flavostatin,Graminelysin I, H2-Proteinase, HF3, HR1A, HR1B, HR2A, HT-2, HV1,Jararhagin, Jerdonitin, Kaouthiagin, Lebatase, LHF-II, MT-D, RVV-X,Trigramin, VAP1, VLFXA (see Fox et al., Toxicon 45 (2005), 969-985).Amino acid sequences and polypeptides of the invention could also bedeveloped as antidotes for such venoms.

The polypeptides and compositions of the present invention can generallybe used to modulate, and in particular inhibit and/or prevent, theinteraction between a metalloproteinase from the ADAM family and itssubstrate (and in particular ADAM-mediated ectodomain-shedding, i.e. theADAM-mediated release of extracellular domains from the substrate) andthus to modulate, and in particular inhibit or prevent, the signallingthat is mediated by a metalloproteinase from the ADAM family and/or byits substrate, to modulate the biological pathways in which ametalloproteinase from the ADAM family and/or its substrate is involved,and/or to modulate the biological mechanisms, responses and effectsassociated with such signalling or these pathways.

As such, the polypeptides and compositions of the present invention canbe used for the prevention and treatment (as defined herein) ofADAM-related diseases and disorders. Generally, “ADAM-related diseasesand disorders” can be defined as diseases and disorders that can beprevented and/or treated, respectively, by suitably administering to asubject in need thereof (i.e. having the disease or disorder or at leastone symptom thereof and/or at risk of attracting or developing thedisease or disorder) of either a polypeptide or composition of theinvention (and in particular, of a pharmaceutically active amountthereof) and/or of a known active principle active against ametalloproteinase from the ADAM family (or against one of itssubstrates, and/or against one of the ligands of such a substrate) or abiological pathway or mechanism in which a metalloproteinase from theADAM family (and/or one of its substrates) is involved (and inparticular, of a pharmaceutically active amount thereof). Examples ofsuch ADAM-related diseases and disorders will be clear to the skilledperson based on the disclosure herein, and for example include thediseases and disorders mentioned herein and in the prior art citedherein.

In particular, the polypeptides and compositions of the presentinvention can be used for the prevention and treatment of ADAM-relateddiseases and disorders which are characterized by excessive and/orunwanted signalling mediated by a substrate of a metalloproteinase fromthe ADAM family (or one of the ligands of such a substrate) or by thepathway(s) in which a metalloproteinase from the ADAM family isinvolved. Examples of such ADAM-related diseases and disorders willagain be clear to the skilled person based on the disclosure herein.

Thus, without being limited thereto, the amino acid sequences andpolypeptides of the invention can for example be used to prevent and/orto treat all diseases and disorders that are currently being preventedor treated with active principles that can modulate a metalloproteinasefrom the ADAM family-mediated signalling, such as those mentioned in theprior art cited above. It is also envisaged that the polypeptides of theinvention can be used to prevent and/or to treat all diseases anddisorders for which treatment with such active principles is currentlybeing developed, has been proposed, or will be proposed or developed infuture. In addition, it is envisaged that, because of their favourableproperties as further described herein, the polypeptides of the presentinvention may be used for the prevention and treatment of other diseasesand disorders than those for which these known active principles arebeing used or will be proposed or developed; and/or that thepolypeptides of the present invention may provide new methods andregimens for treating the diseases and disorders described herein.

Other applications and uses of the amino acid sequences and polypeptidesof the invention will become clear to the skilled person from thefurther disclosure herein.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of ADAM-relateddiseases and disorders and of the further diseases and disordersmentioned herein; and to provide methods for the diagnosis, preventionand/or treatment of such diseases and disorders that involve theadministration and/or use of such agents and compositions.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods that havecertain advantages compared to the agents, compositions and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as compositions comprising the same, for the diagnosis,prevention and/or treatment of ADAM-related diseases and disorders andof the further diseases and disorders mentioned herein; and to providemethods for the diagnosis, prevention and/or treatment of such diseasesand disorders that involve the administration and/or the use of suchtherapeutic proteins and compositions.

Accordingly, it is a specific object of the present invention to provideamino acid sequences that are directed against (as defined herein) ametalloproteinase from the ADAM family, in particular against ametalloproteinase from the ADAM family from a warm-blooded animal, morein particular against a metalloproteinase from the ADAM family from amammal, and especially against human a metalloproteinase from the ADAMfamily; and to provide proteins and polypeptides comprising oressentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat are suitable for prophylactic, therapeutic and/or diagnostic use ina warm-blooded animal, and in particular in a mammal, and more inparticular in a human being.

More in particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat can be used for the prevention, treatment, alleviation and/ordiagnosis of one or more diseases, disorders or conditions associatedwith a metalloproteinase from the ADAM family and/or mediated by ametalloproteinase from the ADAM family (such as the diseases, disordersand conditions mentioned herein) in a warm-blooded animal, in particularin a mammal, and more in particular in a human being.

It is also a specific object of the invention to provide such amino acidsequences and such proteins and/or polypeptides that can be used in thepreparation of pharmaceutical or veterinary compositions for theprevention and/or treatment of one or more diseases, disorders orconditions associated with and/or mediated by a metalloproteinase fromthe ADAM family (such as the diseases, disorders and conditionsmentioned herein) in a warm-blooded animal, in particular in a mammal,and more in particular in a human being.

In the invention, generally, these objects are achieved by the use ofthe amino acid sequences, proteins, polypeptides and compositions thatare described herein.

In general, the invention provides amino acid sequences that aredirected against (as defined herein) and/or can specifically bind (asdefined herein) to a metalloproteinase from the ADAM family; as well ascompounds and constructs, and in particular proteins and polypeptides,that comprise at least one such amino acid sequence.

More in particular, the invention provides amino acid sequences that canbind to a metalloproteinase from the ADAM family with an affinity(suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein; as well as compounds and constructs,and in particular proteins and polypeptides, that comprise at least onesuch amino acid sequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to a metalloproteinase from the ADAM family with a        dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or        less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more        preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association        constant (K_(A)) of 10⁵ to 10¹² liter/ moles or more, and        preferably 10⁷ to 10¹² liter/moles or more and more preferably        10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to a metalloproteinase from the ADAM family with a        k_(on)-rate of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹,        preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻s⁻¹, more preferably        between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹        and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to a metalloproteinase from the ADAM family with a        k_(off)-rate between 1 s^(−1 (t) _(1/2)=0.69 s) and 10⁻⁶ s⁻¹        (providing a near irreversible complex with a t_(1/2) of        multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more        preferably between 10⁻³ s⁻¹ and 10⁻⁴ s⁻¹, such as between 10⁻⁴        s⁻¹ and 10⁻⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to a metalloproteinase from theADAM family with an affinity less than 500 nM, preferably less than 200nM, more preferably less than 10 nM, such as less than 500 pM.

Some preferred IC50 values for binding of the amino acid sequences orpolypeptides of the invention to a metalloproteinase from the ADAMfamily will become clear from the further description and examplesherein.

For binding to a metalloproteinase from the ADAM family, an amino acidsequence of the invention will usually contain within its amino acidsequence one or more amino acid residues or one or more stretches ofamino acid residues (i.e. with each “stretch” comprising two or aminoacid residues that are adjacent to each other or in close proximity toeach other, i.e. in the primary or tertiary structure of the amino acidsequence) via which the amino acid sequence of the invention can bind toa metalloproteinase from the ADAM family, which amino acid residues orstretches of amino acid residues thus form the “site” for binding to ametalloproteinase from the ADAM family (also referred to herein as the“antigen binding site”).

The amino acid sequences provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more amino acid sequences of theinvention and which may optionally further comprise one or more furtheramino acid sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or more aminoacid sequences of the invention may be used as a binding unit in such aprotein or polypeptide, which may optionally contain one or more furtheramino acid sequences that can serve as a binding unit (i.e. against oneor more other targets than a metalloproteinase from the ADAM family), soas to provide a monovalent, multivalent or multispecific polypeptide ofthe invention, respectively, all as described herein. Such a protein orpolypeptide may also be in essentially isolated form (as definedherein).

The amino acid sequences and polypeptides of the invention as suchpreferably essentially consist of a single amino acid chain that is notlinked via disulphide bridges to any other amino acid sequence or chain(but that may or may not contain one or more intramolecular disulphidebridges. For example, it is known that Nanobodies—as describedherein—may sometimes contain a disulphide bridge between CDR3 and CDR1or FR2). However, it should be noted that one or more amino acidsequences of the invention may be linked to each other and/or to otheramino acid sequences (e.g. via disulphide bridges) to provide peptideconstructs that may also be useful in the invention (for example Fab′fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and othermultispecific constructs. Reference is for example made to the review byHolliger and Hudson, Nat Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the amino acid sequences of the invention (as well as compounds,constructs and polypeptides comprising the same) are preferably directedagainst human a metalloproteinase from the ADAM family; whereas forveterinary purposes, the amino acid sequences and polypeptides of theinvention are preferably directed against a metalloproteinase from theADAM family from the species to be treated, or at least cross-reactivewith a metalloproteinase from the ADAM family from the species to betreated.

Furthermore, an amino acid sequence of the invention may optionally, andin addition to the at least one binding site for binding against ametalloproteinase from the ADAM family, contain one or more furtherbinding sites for binding against other antigens, proteins or targets.

The efficacy of the amino acid sequences and polypeptides of theinvention, and of compositions comprising the same, can be tested usingany suitable in vitro assay, cell-based assay, in vivo assay and/oranimal model known per se, or any combination thereof, depending on thespecific disease or disorder involved. Suitable assays and animal modelswill be clear to the skilled person, and for example include techniquessuch as BIACORE and FLIPR; commercially available protease assays (forexample the assays for ADAM-(TS) available from R&D Systems. For ADAM8,9, 10 and 17 the ES003 substrate can be used); cell-based assays thatmeasure sheddase activity (for example for CD23, EGF or TNF, see Weskampet at, supra and the references cited therein), as well as the assaysand animal models used in the experimental part below and in the priorart cited herein. Also, assays can be used that measure the activity ofthe substrate of ADAM or the signalling or biological activities inwhich such a substrate (or its natural ligand) in involved.

For example, TNFalpha release (cleavage) can be induced in monocytes andinactivating or activating ADAM17 modulators can be tested in thissetup. These and similar assays are for example described in Newton etal. Ann Rheum Dis 60 Suppl 3, iii25-32 (2001).

In animal models, ADAM10 activity can be quantified using soluble CD23(see for example Weskamp et at 2006, supra, and the references citedtherein). Antigen induced arthritis model can be used to test ADAM-TS5Nanobodies. (ADAM-TS5−/− mice are more resistant to arthritis in thismodel).

An ELISA-based kit can be purchased from Invitek to test the activity ofamino acid sequences or polypeptides of the invention on ADAM-TS5. Fortesting the activity of of amino acid sequences or polypeptides of theinvention on ADAM10 in a cell-based assay, cleavage of CD44 into themedium of cell can be used to detect the activity of ADAM10 in U251glioblastoma (S. Atkinson and G. Murphy, publication submitted). Fortesting the activity of of amino acid sequences or polypeptides of theinvention on ADAM17 in a cell-based assay, the release of HB-EGFconjugated Alkaline phosphatase from transfected MCF7 cells can be useda readout for ADAM17 activity (S. Atkinson and G. Murphy, submitted).

Also, according to the invention, amino acid sequences and polypeptidesthat are directed against a metalloproteinase from the ADAM family froma first species of warm-blooded animal may or may not showcross-reactivity with a metalloproteinase from the ADAM family from oneor more other species of warm-blooded animal. For example, amino acidsequences and polypeptides directed against human a metalloproteinasefrom the ADAM family may or may not show cross reactivity with ametalloproteinase from the ADAM family from one or more other species ofprimates (such as, without limitation, monkeys from the genus Macaca(such as, and in particular, cynomolgus monkeys (Macaca fascicularis)and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus))and/or with a metalloproteinase from the ADAM family from one or morespecies of animals that are often used in animal models for diseases(for example mouse, rat, rabbit, pig or dog), and in particular inanimal models for diseases and disorders associated with ametalloproteinase from the ADAM family (such as the species and animalmodels mentioned herein). In this respect, it will be clear to theskilled person that such cross-reactivity, when present, may haveadvantages from a drug development point of view, since it allows theamino acid sequences and polypeptides against human a metalloproteinasefrom the ADAM family to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the inventionthat are cross-reactive with a metalloproteinase from the ADAM familyfrom multiple species of mammal will usually be advantageous for use inveterinary applications, since it will allow the same amino acidsequence or polypeptide to be used across multiple species. Thus, it isalso encompassed within the scope of the invention that amino acidsequences and polypeptides directed against a metalloproteinase from theADAM family from one species of animal (such as amino acid sequences andpolypeptides against human a metalloproteinase from the ADAM family) canbe used in the treatment of another species of animal, as long as theuse of the amino acid sequences and/or polypeptides provide the desiredeffects in the species to be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of ametalloproteinase from the ADAM family against which the amino acidsequences and polypeptides of the invention are directed. For example,the amino acid sequences and polypeptides may or may not be directedagainst an “interaction site” (as defined herein). However, somepreferred amino acid sequences of the invention may be directed againstone or more of the following epitopes, domains, active/catalytic sitesor binding sites:

-   -   against the catalytic site: such amino acid sequences may for        example reduce, inhibit or inactivate the protease activity;    -   against the catalytic domain and/or the PRO-domain-catalytic        site (such that it is disrupted): such interaction may for        example activate or increase the protease activity;    -   against the PRO-domain and /or against the PRO-catalytic site        (such that it is disrupted): such interaction may for example        activate or increase the protease activity;    -   against the Disintegrin/cys-rich domain: such interaction may        for example modulate the targeting of the ADAM or its binding to        substrate, or its selectivity for the substrate;    -   against the site on the ADAM proteinase for interaction with        TIMP, thus modulating (and in particular inhibiting) the        regulation of ADAM by TIMPs (TIMP1,2,3 or 4).

Also, generally, some preferred amino acid sequences and polypeptides ofthe invention are preferably such that they are capable of inhibitingthe activity of the ADAM proteinase, as measured by a suitable assay(such as a commercially available protease assay) under conditionsusually applied for such an assay (as will be clear to the skilledperson), by at least 1%, preferably at least 5%, more preferably atleast 10%, such as at least 25% or even more than 50% and up to 75% ormore, such as 90% or more, compared to the activity of the ADAMproteinase without the presence of the amino acid sequence orpolypeptide of the invention.

Also, generally, some preferred amino acid sequences and polypeptides ofthe invention are preferably such that they are capable of activating(the protease activity of) the ADAM proteinase, as measured by asuitable assay (such as a commercially available protease assay) underconditions usually applied for such an assay (as will be clear to theskilled person), by at least 1%, preferably at least 5%, more preferablyat least 10%, such as at least 25% or even more than 50% and up to 75%or more, such as 90% or more, compared to the activity of the ADAMproteinase without the presence of the amino acid sequence orpolypeptide of the invention.

Some other preferred amino acid sequences and polypeptides may be suchthat they are capable of competing for binding to the ADAM proteinasewith the usual substrate of the ADAM proteinase, and/or with a substratefor an ADAM proteinase that is usually used as a model substrate in anassay for determining the activity of said proteinase.

Some other preferred amino acid sequences and polypeptides may be suchthat they are capable of modulating (as defined herein) the regulationof the ADAM proteinase by TIMP. As is known in the art, therapiesinvolving the use of TIMP or targeting may lead to an undesirableinhibition of metalloproteases, which may cause side effects. Thus, oneaspect of the invention relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention, or of a composition comprisingthe same (methods or compositions for) preventing or reducing the sideeffects of TIMP-dependent therapies or therapies directed against ormediated by TIMP; and to uses of the amino acid sequences, Nanobodiesand polypeptides of the invention in the preparation of pharmaceuticalcompositions for reducing the side effects of TIMP-dependent therapiesor therapies directed against or mediated by TIMP; or with such reducedside effects.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention, or of a composition comprisingthe same, in (methods or compositions for) modulating (as definedherein) an ADAM metalloproteinase, either in vitro (e.g. in an in vitroor cellular assay) or in vivo (e.g. in an a single cell or in amulticellular organism, and in particular in a mammal, and more inparticular in a human being, such as in a human being that is at risk ofor suffers from ADAM-related diseases and disorders).

The invention also relates to methods for modulating (as defined herein)an ADAM metalloproteinase, either in vitro (e.g. in an in vitro orcellular assay) or in vivo (e.g. in an a single cell or multicellularorganism, and in particular in a mammal, and more in particular in ahuman being, such as in a human being that is at risk of or suffers fromADAM-related diseases and disorders), which method comprises at leastthe step of contacting an ADAM metalloproteinase with at least one aminoacid sequence, Nanobody or polypeptide of the invention, or with acomposition comprising the same, in a manner and in an amount suitableto modulate an ADAM metalloproteinase, with at least one amino acidsequence, Nanobody or polypeptide of the invention.

The invention also relates to the use of an one amino acid sequence,Nanobody or polypeptide of the invention in the preparation of acomposition (such as, without limitation, a pharmaceutical compositionor preparation as further described herein) for modulating (as definedherein) an ADAM metalloproteinase, either in vitro (e.g. in an in vitroor cellular assay) or in vivo (e.g. in an a single cell or multicellularorganism, and in particular in a mammal, and more in particular in ahuman being, such as in a human being that is at risk of or suffers froma ADAM-related diseases and disorders).

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be in the prevention and treatment of sepsis. Thus, furtheraspects of the invention relate to the use of an amino acid sequence,Nanobody or polypeptide of the invention, or of a composition comprisingthe same, in (methods or compositions for) the treatment (as definedherein) of sepsis; to pharmaceutical compositions and preparations (asdescribed herein) for the treatment of sepsis; and to the use of theamino acid sequences, Nanobodies and polypeptides of the invention inmethods for preparing pharmaceutical compositions and preparations (asdescribed herein) for the treatment of sepsis.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be used in the prevention and treatment of the side effects ofTIMP-related therapies. Thus, further aspects of the invention relate tothe use of an amino acid sequence, Nanobody or polypeptide of theinvention, or of a composition comprising the same, in (methods orcompositions for) the prevention and/or treatment (as defined herein) ofthe side effects of TIMP-related therapies; to pharmaceuticalcompositions and preparations (as described herein) for the preventionand/or treatment (as defined herein) of the side effects of TIMP-relatedtherapies; and to the use of the amino acid sequences, Nanobodies andpolypeptides of the invention in methods for preparing pharmaceuticalcompositions and preparations (as described herein) for the preventionand/or treatment (as defined herein) of the side effects of TIMP-relatedtherapies.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be used to inhibit or reduce the release of one or more growthfactors, for example in the treatment of cancer. Thus, further aspectsof the invention relate to the use of an amino acid sequence, Nanobodyor polypeptide of the invention, or of a composition comprising thesame, in (methods or compositions for) inhibiting or reducing therelease of one or more growth factors, for example in the treatment ofcancer; to pharmaceutical compositions and preparations (as describedherein) for inhibiting or reducing the release of one or more growthfactors, for example in the treatment of cancer; and to the use of theamino acid sequences, Nanobodies and polypeptides of the invention inmethods for preparing pharmaceutical compositions and preparations (asdescribed herein) for inhibiting or reducing the release of one or moregrowth factors, for example in the treatment of cancer.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be used to inhibit or reduce the release of one or morecytokines, for example in the prevention or treatment of inflammation.Thus, further aspects of the invention relate to the use of an aminoacid sequence, Nanobody or polypeptide of the invention, or of acomposition comprising the same, in (methods or compositions for)inhibiting or reducing the release of one or more cytokines, for examplein the prevention or treatment of inflammation; to pharmaceuticalcompositions and preparations (as described herein) for inhibiting orreducing the release of one or more cytokines, for example in theprevention or treatment of inflammation; and to the use of the aminoacid sequences, Nanobodies and polypeptides of the invention in methodsfor preparing pharmaceutical compositions and preparations (as describedherein) for inhibiting or reducing the release of one or more cytokines,for example in the prevention or treatment of inflammation.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be used as a marker for cells that express ADAM proteinases(such as ADAM15) on their surface, for example for detecting tumourcells or atherosclerosis, for example in vitro or by means of in vivoimaging. Reference is made to the further disclosure herein.

As further described herein, a polypeptide of the invention may containtwo or more amino acid sequences of the invention that are directedagainst an ADAM proteinase. Generally, such polypeptides will bind tothe ADAM proteinase with increased avidity compared to a single aminoacid sequence of the invention. Such a polypeptide may for examplecomprise two amino acid sequences of the invention that are directedagainst the same antigenic determinant, epitope, part, domain, subunitor confirmation (where applicable) of ADAM proteinase (which may or maynot be an interaction site); or comprise at least one “first” amino acidsequence of the invention that is directed against a first sameantigenic determinant, epitope, part, domain, subunit or confirmation(where applicable) of ADAM proteinase (which may or may not be aninteraction site); and at least one “second” amino acid sequence of theinvention that is directed against a second antigenic determinant,epitope, part, domain, subunit or confirmation (where applicable)different from the first (and which again may or may not be aninteraction site). Preferably, in such “biparatopic” polypeptides of theinvention, at least one amino acid sequence of the invention is directedagainst an interaction site (as defined herein), although the inventionin its broadest sense is not limited thereto.

Also, when the target is part of a binding pair (for example, areceptor-ligand binding pair), the amino acid sequences and polypeptidesmay be such that they compete with the cognate binding partner (e.g. theligand, receptor or other binding partner, as applicable) for binding tothe target, and/or such that they (fully or partially) neutralizebinding of the binding partner to the target.

It is also within the scope of the invention that, where applicable, anamino acid sequence of the invention can bind to two or more antigenicdeterminants, epitopes, parts, domains, subunits or confirmations of ametalloproteinase from the ADAM family. In such a case, the antigenicdeterminants, epitopes, parts, domains or subunits of ametalloproteinase from the ADAM family to which the amino acid sequencesand/or polypeptides of the invention bind may be essentially the same(for example, if a metalloproteinase from the ADAM family containsrepeated structural motifs or occurs in a multimeric form) or may bedifferent (and in the latter case, the amino acid sequences andpolypeptides of the invention may bind to such different antigenicdeterminants, epitopes, parts, domains, subunits of a metalloproteinasefrom the ADAM family with an affinity and/or specificity which may bethe same or different). Also, for example, when a metalloproteinase fromthe ADAM family exists in an activated conformation and in an inactiveconformation, the amino acid sequences and polypeptides of the inventionmay bind to either one of these confirmation, or may bind to both theseconfirmations (i.e. with an affinity and/or specificity which may be thesame or different). Also, for example, the amino acid sequences andpolypeptides of the invention may bind to a conformation of ametalloproteinase from the ADAM family in which it is bound to apertinent ligand, may bind to a conformation of a metalloproteinase fromthe ADAM family in which it not bound to a pertinent ligand, or may bindto both such conformations (again with an affinity and/or specificitywhich may be the same or different).

It is also expected that the amino acid sequences and polypeptides ofthe invention will generally bind to all naturally occurring orsynthetic analogs, variants, mutants, alleles, parts and fragments of ametalloproteinase from the ADAM family; or at least to those analogs,variants, mutants, alleles, parts and fragments of a metalloproteinasefrom the ADAM family that contain one or more antigenic determinants orepitopes that are essentially the same as the antigenic determinant(s)or epitope(s) to which the amino acid sequences and polypeptides of theinvention bind in a metalloproteinase from the ADAM family (e.g. inwild-type a metalloproteinase from the ADAM family). Again, in such acase, the amino acid sequences and polypeptides of the invention maybind to such analogs, variants, mutants, alleles, parts and fragmentswith an affinity and/or specificity that are the same as, or that aredifferent from (i.e. higher than or lower than), the affinity andspecificity with which the amino acid sequences of the invention bind to(wild-type) a metalloproteinase from the ADAM family. It is alsoincluded within the scope of the invention that the amino acid sequencesand polypeptides of the invention bind to some analogs, variants,mutants, alleles, parts and fragments of a metalloproteinase from theADAM family, but not to others.

When a metalloproteinase from the ADAM family exists in a monomeric formand in one or more multimeric forms, it is within the scope of theinvention that the amino acid sequences and polypeptides of theinvention only bind to a metalloproteinase from the ADAM family inmonomeric form, only bind to a metalloproteinase from the ADAM family inmultimeric form, or bind to both the monomeric and the multimeric form.Again, in such a case, the amino acid sequences and polypeptides of theinvention may bind to the monomeric form with an affinity and/orspecificity that are the same as, or that are different from (i.e.higher than or lower than), the affinity and specificity with which theamino acid sequences of the invention bind to the multimeric form.

Also, when a metalloproteinase from the ADAM family can associate withother proteins or polypeptides to form protein complexes (e.g. withmultiple subunits), it is within the scope of the invention that theamino acid sequences and polypeptides of the invention bind to ametalloproteinase from the ADAM family in its non-associated state, bindto a metalloproteinase from the ADAM family in its associated state, orbind to both. In all these cases, the amino acid sequences andpolypeptides of the invention may bind to such multimers or associatedprotein complexes with an affinity and/or specificity that may be thesame as or different from (i.e. higher than or lower than) the affinityand/or specificity with which the amino acid sequences and polypeptidesof the invention bind to a metalloproteinase from the ADAM family in itsmonomeric and non-associated state.

Also, as will be clear to the skilled person, proteins or polypeptidesthat contain two or more amino acid sequences directed against ametalloproteinase from the ADAM family may bind with higher avidity to ametalloproteinase from the ADAM family than the corresponding monomericamino acid sequence(s). For example, and without limitation, proteins orpolypeptides that contain two or more amino acid sequences directedagainst different epitopes of a metalloproteinase from the ADAM familymay (and usually will) bind with higher avidity than each of thedifferent monomers, and proteins or polypeptides that contain two ormore amino acid sequences directed against a metalloproteinase from theADAM family may (and usually will) bind also with higher avidity to amultimer of a metalloproteinase from the ADAM family.

Generally, amino acid sequences and polypeptides of the invention willat least bind to those forms of a metalloproteinase from the ADAM family(including monomeric, multimeric and associated forms) that are the mostrelevant from a biological and/or therapeutic point of view, as will beclear to the skilled person.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the amino acidsequences and polypeptides of the invention, and/or to use proteins orpolypeptides comprising or essentially consisting of one or more of suchparts, fragments, analogs, mutants, variants, alleles and/orderivatives, as long as these are suitable for the uses envisagedherein. Such parts, fragments, analogs, mutants, variants, allelesand/or derivatives will usually contain (at least part of) a functionalantigen-binding site for binding against a metalloproteinase from theADAM family; and more preferably will be capable of specific binding toa metalloproteinase from the ADAM family, and even more preferablycapable of binding to a metalloproteinase from the ADAM family with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Some non-limiting examples of suchparts, fragments, analogs, mutants, variants, alleles, derivatives,proteins and/or polypeptides will become clear from the furtherdescription herein. Additional fragments or polypeptides of theinvention may also be provided by suitably combining (i.e. by linking orgenetic fusion) one or more (smaller) parts or fragments as describedherein.

In one specific, but non-limiting aspect of the invention, which will befurther described herein, such analogs, mutants, variants, alleles,derivatives have an increased half-life in serum (as further describedherein) compared to the amino acid sequence from which they have beenderived. For example, an amino acid sequence of the invention may belinked (chemically or otherwise) to one or more groups or moieties thatextend the half-life (such as PEG), so as to provide a derivative of anamino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or may be an amino acid sequence that, under suitable conditions(such as physiological conditions) is capable of forming animmunoglobulin fold (i.e. by folding). Reference is inter alia made tothe review by Halaby et al., J. (1999) Protein Eng. 12, 563-71.Preferably, when properly folded so as to form an immunoglobulin fold,such an amino acid sequence is capable of specific binding (as definedherein) to a metalloproteinase from the ADAM family; and more preferablycapable of binding to a metalloproteinase from the ADAM family with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Also, parts, fragments, analogs,mutants, variants, alleles and/or derivatives of such amino acidsequences are preferably such that they comprise an immunoglobulin foldor are capable for forming, under suitable conditions, an immunoglobulinfold.

In particular, but without limitation, the amino acid sequences of theinvention may be amino acid sequences that essentially consist of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively); or any suitablefragment of such an amino acid sequence (which will then usually containat least some of the amino acid residues that form at least one of theCDR's, as further described herein).

The amino acid sequences of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin variable domain sequence or a suitablefragment thereof, such as light chain variable domain sequence (e.g. aV_(L)-sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H)-sequence) or a suitable fragmentthereof. When the amino acid sequence of the invention is a heavy chainvariable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called V_(HH)-sequence (as defined herein) that isderived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to theorigin of the amino acid sequence of the invention (or of the nucleotidesequence of the invention used to express it), nor as to the way thatthe amino acid sequence or nucleotide sequence of the invention is (orhas been) generated or obtained. Thus, the amino acid sequences of theinvention may be naturally occurring amino acid sequences (from anysuitable species) or synthetic or semi-synthetic amino acid sequences.In a specific but non-limiting aspect of the invention, the amino acidsequence is a naturally occurring immunoglobulin sequence (from anysuitable species) or a synthetic or semi-synthetic immunoglobulinsequence, including but not limited to “humanized” (as defined herein)immunoglobulin sequences (such as partially or fully humanized mouse orrabbit immunoglobulin sequences, and in particular partially or fullyhumanized V_(HH) sequences or Nanobodies), “camelized” (as definedherein) immunoglobulin sequences, as well as immunoglobulin sequencesthat have been obtained by techniques such as affinity maturation (forexample, starting from synthetic, random or naturally occurringimmunoglobulin sequences), CDR grafting, veneering, combining fragmentsderived from different immunoglobulin sequences, PCR assembly usingoverlapping primers, and similar techniques for engineeringimmunoglobulin sequences well known to the skilled person; or anysuitable combination of any of the foregoing. Reference is for examplemade to the standard handbooks, as well as to the further descriptionand prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may for example be sequences that are isolated by PCR from asuitable naturally occurring template (e.g. DNA or RNA isolated from acell), nucleotide sequences that have been isolated from a library (andin particular, an expression library), nucleotide sequences that havebeen prepared by introducing mutations into a naturally occurringnucleotide sequence (using any suitable technique known per se, such asmismatch PCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), a single domain antibody (or an amino acid sequence that issuitable for use as a single domain antibody), a “dAb” (or an amino acidsequence that is suitable for use as a dAb) or a Nanobody™ (as definedherein, and including but not limited to a V_(HH) sequence); othersingle variable domains, or any suitable fragment of any one thereof.For a general description of (single) domain antibodies, reference isalso made to the prior art cited above, as well as to EP 0 368 684. Forthe term “dAb's”, reference is for example made to Ward et al. (Nature1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol.,2003, 21(11):484-490; as well as to for example WO 06/030220, WO06/003388 and other published patent applications of Domantis Ltd. Itshould also be noted that, although less preferred in the context of thepresent invention because they are not of mammalian origin, singledomain antibodies or single variable domains can be derived from certainspecies of shark (for example, the so-called “IgNAR domains”, see forexample WO 05/18629).

In particular, the amino acid sequence of the invention may be aNanobody™ (as defined herein) or a suitable fragment thereof. [Note:Nanobody®, Nanobodies® and Nanoclone® are registered trademarks ofAblynx N.V.] Such Nanobodies directed against a metalloproteinase fromthe ADAM family will also be referred to herein as “Nanobodies of theinvention”.

For a general description of Nanobodies, reference is made to thefurther description below, as well as to the prior art cited herein. Inthis respect, it should however be noted that this description and theprior art mainly described Nanobodies of the so-called “V_(H)3 class”(i.e. Nanobodies with a high degree of sequence homology to humangermline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29),which Nanobodies form a preferred aspect of this invention. It shouldhowever be noted that the invention in its broadest sense generallycovers any type of Nanobody directed against a metalloproteinase fromthe ADAM family, and for example also covers the Nanobodies belonging tothe so-called “V_(H)4 class” (i.e. Nanobodies with a high degree ofsequence homology to human germline sequences of the V_(H)4 class suchas DP-78), as for example described in the U.S. provisional application60/792,279 by Ablynx N.V. entitled “DP-78-like Nanobodies” filed on Apr.14, 2006 (see also PCT/EP2007/003259).

Generally, Nanobodies (in particular V_(HH) sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” (as described herein) in one or moreof the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as an amino acid sequencewith the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which one or more of the Hallmark residues are as further        defined herein.

In particular, a Nanobody can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which the framework sequences are as further defined herein.

More in particular, a Nanobody can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   a) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below;    and in which:

-   b) said amino acid sequence has at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 1 to    22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Thus, the invention also relates to such Nanobodies that can bind to (asdefined herein) and/or are directed against a metalloproteinase from theADAM family, to suitable fragments thereof, as well as to polypeptidesthat comprise or essentially consist of one or more of such Nanobodiesand/or suitable fragments.

SEQ ID NO's 868 to 973 and 1044 to 1053 give the amino acid sequences ofa number of V_(HH) sequences that have been raised against ametalloproteinase from the ADAM family, i.e.:

-   -   SEQ ID NO's 868 to 886: Nanobodies against ADAM8    -   SEQ ID NO's 887 to 907 and 1044 to 1047: Nanobodies against        ADAM9    -   SEQ ID NO's 908 to 931: Nanobodies against ADAM10    -   SEQ ID NO's 932 to 948 and 1048 to 1052: Nanobodies against        ADAM17    -   SEQ ID NO's 949 to 974 and 1053: Nanobodies against ADAMTS5

In particular, the invention in some specific aspects provides:

-   -   amino acid sequences that are directed against (as defined        herein) ADAM8 and that have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 868 to 886.        These amino acid sequences may further be such that they        neutralize binding of the cognate ligand to ADAM8; and/or        compete with the cognate ligand for binding to ADAM8; and/or are        directed against an interaction site (as defined herein) on        ADAM8 (such as the ligand binding site);    -   amino acid sequences that cross-block (as defined herein) the        binding of at least one of the amino acid sequences of SEQ ID        NO's: 868 to 886 to ADAM8 and/or that compete with at least one        of the amino acid sequences of SEQ ID NO's: 868 to 886 for        binding to ADAM8. Again, these amino acid sequences may further        be such that they neutralize binding of the cognate ligand to        ADAM8; and/or compete with the cognate ligand for binding to        ADAM8; and/or are directed against an interaction site (as        defined herein) on ADAM8 (such as the ligand binding site);    -   amino acid sequences that are directed against (as defined        herein) ADAM9 and that have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 887 to 907 and        1044 to 1047. These amino acid sequences may further be such        that they neutralize binding of the cognate ligand to ADAM9;        and/or compete with the cognate ligand for binding to ADAM9;        and/or are directed against an interaction site (as defined        herein) on ADAM9 (such as the ligand binding site);    -   amino acid sequences that cross-block (as defined herein) the        binding of at least one of the amino acid sequences of SEQ ID        NO's: 887 to 907 and 1044 to 1047to ADAM9 and/or that compete        with at least one of the amino acid sequences of SEQ ID NO's:        887 to 907 and 1044 to 1047 for binding to ADAM9. Again, these        amino acid sequences may further be such that they neutralize        binding of the cognate ligand to ADAM9; and/or compete with the        cognate ligand for binding to ADAM9; and/or are directed against        an interaction site (as defined herein) on ADAM9 (such as the        ligand binding site);    -   amino acid sequences that are directed against (as defined        herein) ADAM10 and that have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 908 to 931.        These amino acid sequences may further be such that they        neutralize binding of the cognate ligand to ADAM10; and/or        compete with the cognate ligand for binding to ADAM10; and/or        are directed against an interaction site (as defined herein) on        ADAM10 (such as the ligand binding site);    -   amino acid sequences that cross-block (as defined herein) the        binding of at least one of the amino acid sequences of SEQ ID        NO's: 908 to 931to ADAM10 and/or that compete with at least one        of the amino acid sequences of SEQ ID NO's: 908 to 931 for        binding to ADAM10. Again, these amino acid sequences may further        be such that they neutralize binding of the cognate ligand to        ADAM10; and/or compete with the cognate ligand for binding to        ADAM10; and/or are directed against an interaction site (as        defined herein) on ADAM10 (such as the ligand binding site);    -   amino acid sequences that are directed against (as defined        herein) ADAM17 and that have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 932 to 948 and        1048 to 1052. These amino acid sequences may further be such        that they neutralize binding of the cognate ligand to ADAM17;        and/or compete with the cognate ligand for binding to ADAM17;        and/or are directed against an interaction site (as defined        herein) on ADAM 17 (such as the ligand binding site);    -   amino acid sequences that cross-block (as defined herein) the        binding of at least one of the amino acid sequences of SEQ ID        NO's: 932 to 948 and 1048 to 1052to ADAM17 and/or that compete        with at least one of the amino acid sequences of SEQ ID NO's:        932 to 948 and 1048 to 1052 for binding to ADAM17. Again, these        amino acid sequences may further be such that they neutralize        binding of the cognate ligand to ADAM17; and/or compete with the        cognate ligand for binding to ADAM17; and/or are directed        against an interaction site (as defined herein) on ADAM 17 (such        as the ligand binding site);    -   amino acid sequences that are directed against (as defined        herein) ADAMTS5 and that have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 949 to 974        and 1053. These amino acid sequences may further be such that        they neutralize binding of the cognate ligand to ADAMTS5; and/or        compete with the cognate ligand for binding to ADAMTS5; and/or        are directed against an interaction site (as defined herein) on        ADAMTS5 (such as the ligand binding site);    -   amino acid sequences that cross-block (as defined herein) the        binding of at least one of the amino acid sequences of SEQ ID        NO's: 949 to 974 and 1053 to ADAMTS5 and/or that compete with at        least one of the amino acid sequences of SEQ ID NO's: 949 to 974        and 1053 for binding to ADAMTS5. Again, these amino acid        sequences may further be such that they neutralize binding of        the cognate ligand to ADAMTS5; and/or compete with the cognate        ligand for binding to ADAMTS5; and/or are directed against an        interaction site (as defined herein) on ADAMTS5 (such as the        ligand binding site);        which amino acid sequences may be as further described herein        (and may for example be Nanobodies); as well as polypeptides of        the invention that comprise one or more of such amino acid        sequences (which may be as further described herein, and may for        example be bispecific and/or biparatopic polypeptides as        described herein), and nucleic acid sequences that encode such        amino acid sequences and polypeptides. Such amino acid sequences        and polypeptides do not include any naturally occurring ligands.

In some other specific aspects, the invention provides:

-   -   amino acid sequences of the invention that are specific for (as        defined herein) ADAM8 compared to ADAM9, ADAM10, ADAM17 and/or        ADAMTS5. These preferably have have at least 80%, preferably at        least 85%, such as 90% or 95% or more sequence identity with at        least one of the amino acid sequences of SEQ ID NO's: 868 to        886;    -   amino acid sequences of the invention that are specific for (as        defined herein) ADAM9 compared to ADAM8, ADAM10, ADAM17 and/or        ADAMTS5. These preferably have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 887 to 907 and        1044 to 1047.    -   amino acid sequences of the invention that are specific for (as        defined herein) ADAM10 compared to ADAM8, ADAM9, ADAM17 and/or        ADAMTS5. These preferably have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 908 to 931;    -   amino acid sequences of the invention that are specific for (as        defined herein) ADAM17 compared to ADAM8, ADAM9, ADAM10 and/or        ADAMTS5. These preferably have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 932 to 948 and        1048 to 1052;    -   amino acid sequences of the invention that are specific for (as        defined herein) ADAMTS5 compared to ADAM8, ADAM9, ADAM10 and/or        ADAM17. These preferably have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 949 to 974 and        1053;        which amino acid sequences of the invention may be as further        described herein (and may for example be Nanobodies); as well as        polypeptides of the invention that comprise one or more of such        amino acid sequences (which may be as further described herein,        and may for example be bispecific and/or biparatopic        polypeptides as described herein), and nucleic acid sequences        that encode such amino acid sequences and polypeptides. Such        amino acid sequences and polypeptides do not include any        naturally occurring ligands.

Accordingly, some particularly preferred Nanobodies of the invention areNanobodies which can bind (as further defined herein) to and/or aredirected against to a metalloproteinase from the ADAM family and which:

-   a) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053,    in which for the purposes of determining the degree of amino acid    identity, the amino acid residues that form the CDR sequences are    disregarded. In this respect, reference is also made to Table A-1,    which lists the framework 1 sequences (SEQ ID NO's: 126 to 231),    framework 2 sequences (SEQ ID NO's: 338 to 443), framework 3    sequences (SEQ ID NO's: 550 to 655) and framework 4 sequences (SEQ    ID NO's: 762 to 867) of the Nanobodies of SEQ ID NO's: 868 to 973    and/or 1044 to 1053 (with respect to the amino acid residues at    positions 1 to 4 and 27 to 30 of the framework 1 sequences,    reference is also made to the comments made below. Thus, for    determining the degree of amino acid identity, these residues are    preferably disregarded);    and in which:-   b) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Again, such Nanobodies may be derived in any suitable manner and fromany suitable source, and may for example be naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences, including but not limited to“humanized” (as defined herein) Nanobodies, “camelized” (as definedherein) immunoglobulin sequences (and in particular camelized heavychain variable domain sequences), as well as Nanobodies that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein. Also, when a Nanobodycomprises a V_(HH) sequence, said Nanobody may be suitably humanized, asfurther described herein, so as to provide one or more further(partially or fully) humanized Nanobodies of the invention. Similarly,when a Nanobody comprises a synthetic or semi-synthetic sequence (suchas a partially humanized sequence), said Nanobody may optionally befurther suitably humanized, again as described herein, again so as toprovide one or more further (partially or fully) humanized Nanobodies ofthe invention.

In particular, humanized Nanobodies may be amino acid sequences that areas generally defined for Nanobodies in the previous paragraphs, but inwhich at least one amino acid residue is present (and in particular, inat least one of the framework residues) that is and/or that correspondsto a humanizing substitution (as defined herein). Some preferred, butnon-limiting humanizing substitutions (and suitable combinationsthereof) will become clear to the skilled person based on the disclosureherein. In addition, or alternatively, other potentially usefulhumanizing substitutions can be ascertained by comparing the sequence ofthe framework regions of a naturally occurring V_(HH) sequence with thecorresponding framework sequence of one or more closely related humanV_(H) sequences, after which one or more of the potentially usefulhumanizing substitutions (or combinations thereof) thus determined canbe introduced into said V_(HH) sequence (in any manner known per se, asfurther described herein) and the resulting humanized V_(HH) sequencescan be tested for affinity for the target, for stability, for ease andlevel of expression, and/or for other desired properties. In this way,by means of a limited degree of trial and error, other suitablehumanizing substitutions (or suitable combinations thereof) can bedetermined by the skilled person based on the disclosure herein. Also,based on the foregoing, (the framework regions of) a Nanobody may bepartially humanized or fully humanized.

Some particularly preferred humanized Nanobodies of the invention arehumanized variants of the Nanobodies of SEQ ID NO's: 868 to 973 and/or1044 to 1053.

Thus, some other preferred Nanobodies of the invention are Nanobodieswhich can bind (as further defined herein) to a metalloproteinase fromthe ADAM family and which:

-   a) are a humanized variant of one of the amino acid sequences of SEQ    ID NO's: 868 to 973 and/or 1044 to 1053; and/or-   b) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053,    in which for the purposes of determining the degree of amino acid    identity, the amino acid residues that form the CDR sequences are    disregarded;    and in which:-   c) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

According to another specific aspect of the invention, the inventionprovides a number of stretches of amino acid residues (i.e. smallpeptides) that are particularly suited for binding to ametalloproteinase from the ADAM family. These stretches of amino acidresidues may be present in, and/or may be corporated into, an amino acidsequence of the invention, in particular in such a way that they form(part of) the antigen binding site of an amino acid sequence of theinvention. As these stretches of amino acid residues were firstgenerated as CDR sequences of heavy chain antibodies or V_(HH) sequencesthat were raised against a metalloproteinase from the ADAM family (ormay be based on and/or derived from such CDR sequences, as furtherdescribed herein), they will also generally be referred to herein as“CDR sequences” (i.e. as CDR1 sequences, CDR2 sequences and CDR3sequences, respectively). It should however be noted that the inventionin its broadest sense is not limited to a specific structural role orfunction that these stretches of amino acid residues may have in anamino acid sequence of the invention, as long as these stretches ofamino acid residues allow the amino acid sequence of the invention tobind to a metalloproteinase from the ADAM family. Thus, generally, theinvention in its broadest sense comprises any amino acid sequence thatis capable of binding to a metalloproteinase from the ADAM family andthat comprises one or more CDR sequences as described herein, and inparticular a suitable combination of two or more such CDR sequences,that are suitably linked to each other via one or more further aminoacid sequences, such that the entire amino acid sequence forms a bindingdomain and/or binding unit that is capable of binding to ametalloproteinase from the ADAM family. It should however also be notedthat the presence of only one such CDR sequence in an amino acidsequence of the invention may by itself already be sufficient to providean amino acid sequence of the invention that is capable of binding to ametalloproteinase from the ADAM family; reference is for example againmade to the so-called “Expedite fragments” described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acidsequence of the invention may be an amino acid sequence that comprisesat least one amino acid sequence that is chosen from the groupconsisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences thatare described herein (or any suitable combination thereof). Inparticular, an amino acid sequence of the invention may be an amino acidsequence that comprises at least one antigen binding site, wherein saidantigen binding site comprises at least one amino acid sequence that ischosen from the group consisting of the CDR1 sequences, CDR2 sequencesand CDR3 sequences that are described herein (or any suitablecombination thereof).

Generally, in this aspect of the invention, the amino acid sequence ofthe invention may be any amino acid sequence that comprises at least onestretch of amino acid residues, in which said stretch of amino acidresidues has an amino acid sequence that corresponds to the sequence ofat least one of the CDR sequences described herein. Such an amino acidsequence may or may not comprise an immunoglobulin fold. For example,and without limitation, such an amino acid sequence may be a suitablefragment of an immunoglobulin sequence that comprises at least one suchCDR sequence, but that is not large enough to form a (complete)immunoglobulin fold (reference is for example again made to the“Expedite fragments” described in WO 03/050531). Alternatively, such anamino acid sequence may be a suitable “protein scaffold” that comprisesleast one stretch of amino acid residues that corresponds to such a CDRsequence (i.e. as part of its antigen binding site). Suitable scaffoldsfor presenting amino acid sequences will be clear to the skilled person,and for example comprise, without limitation, to binding scaffolds basedon or derived from immunoglobulins (i.e. other than the immunoglobulinsequences already described herein), protein scaffolds derived fromprotein A domains (such as Affibodies™), tendamistat, fibronectin,lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimersand PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), andbinding moieties based on DNA or RNA including but not limited to DNA orRNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 20069(8):619-32).

Again, any amino acid sequence of the invention that comprises one ormore of these CDR sequences is preferably such that it can specificallybind (as defined herein) to a metalloproteinase from the ADAM family,and more in particular such that it can bind to a metalloproteinase fromthe ADAM family with an affinity (suitably measured and/or expressed asa K_(D)-value (actual or apparent), a K_(A) value (actual or apparent),a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value,as further described herein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect ofthe invention may be any amino acid sequence that comprises at least oneantigen binding site, wherein said antigen binding site comprises atleast two amino acid sequences that are chosen from the group consistingof the CDR1 sequences described herein, the CDR2 sequences describedherein and the CDR3 sequences described herein, such that (i) when thefirst amino acid sequence is chosen from the CDR1 sequences describedherein, the second amino acid sequence is chosen from the CDR2 sequencesdescribed herein or the CDR3 sequences described herein; (ii) when thefirst amino acid sequence is chosen from the CDR2 sequences describedherein, the second amino acid sequence is chosen from the CDR1 sequencesdescribed herein or the CDR3 sequences described herein; or (iii) whenthe first amino acid sequence is chosen from the CDR3 sequencesdescribed herein, the second amino acid sequence is chosen from the CDR1sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention maybe amino acid sequences that comprise at least one antigen binding site,wherein said antigen binding site comprises at least three amino acidsequences that are chosen from the group consisting of the CDR1sequences described herein, the CDR2 sequences described herein and theCDR3 sequences described herein, such that the first amino acid sequenceis chosen from the CDR1 sequences described herein, the second aminoacid sequence is chosen from the CDR2 sequences described herein, andthe third amino acid sequence is chosen from the CDR3 sequencesdescribed herein. Preferred combinations of CDR1, CDR2 and CDR3sequences will become clear from the further description herein. As willbe clear to the skilled person, such an amino acid sequence ispreferably an immunoglobulin sequence (as further described herein), butit may for example also be any other amino acid sequence that comprisesa suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates toan amino acid sequence directed against a metalloproteinase from theADAM family, that comprises one or more stretches of amino acid residueschosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 232 to 337;-   ii) the amino acid sequences of SEQ ID NO's: 444 to 549; and-   iii) the amino acid sequences of SEQ ID NO's: 656 to 761;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against a metalloproteinase from the ADAM family.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against a metalloproteinase from theADAM family, that comprises two or more stretches of amino acid residueschosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;-   b) the amino acid sequences of SEQ ID NO's: 444 to 549; and-   c) the amino acid sequences of SEQ ID NO's: 656 to 761;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 232    to 337, the second stretch of amino acid residues corresponds to one    of the amino acid sequences of SEQ ID NO's: 444 to 549 or of SEQ ID    NO's: 656 to 761; (ii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 444    to 549, the second stretch of amino acid residues corresponds to one    of the amino acid sequences of SEQ ID NO's: 232 to 337 or of SEQ ID    NO's: 656 to 761; or (iii) when the first stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 656 to 761, the second stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 232    to 337 or of SEQ ID NO's: 444 to 549.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against a metalloproteinase from the ADAM family.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against a metalloproteinase from theADAM family, that comprises three or more stretches of amino acidresidues, in which the first stretch of amino acid residues is chosenfrom the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 232 to 337; the second stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 444 to 549; and the third stretch of amino acid residues is chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's:656 to 761.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against a metalloproteinase from the ADAM family.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 868 to 973 and/or 1044 to 1053. This degree of amino acid identitycan for example be determined by determining the degree of amino acididentity (in a manner described herein) between said amino acid sequenceand one or more of the sequences of SEQ ID NO's: 868 to 973 and/or 1044to 1053, in which the amino acid residues that form the frameworkregions are disregarded. Also, such amino acid sequences of theinvention can be as further described herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to a metalloproteinase from theADAM family; and more in particular bind to a metalloproteinase from theADAM family with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 232 to 337; and/or CDR2 is chosen from thegroup consisting of the amino acid sequences of SEQ ID NO's: 444 to 549;and/or CDR3 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 656 to 761.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 232 to 337; and CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 444 to 549; andCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 656 to 761.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to a metalloproteinase from theADAM family; and more in particular bind to a metalloproteinase from theADAM family with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053.This degree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and one or more of thesequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053, in which theamino acid residues that form the framework regions are disregarded.Such amino acid sequences of the invention can be as further describedherein.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM8 (such as a Nanobody ofthe invention, as further described herein), that comprises one or morestretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;-   b) the amino acid sequences of SEQ ID NO's: 444 to 462; and-   c) the amino acid sequences of SEQ ID NO's: 656 to 674;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against ADAM8.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM8, that comprises two ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;-   b) the amino acid sequences of SEQ ID NO's: 444 to 462; and-   c) the amino acid sequences of SEQ ID NO's: 656 to 674;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 232    to 250, the second stretch of amino acid residues corresponds to one    of the amino acid sequences of SEQ ID NO's: 444 to 462 or of SEQ ID    NO's: 656 to 674; (ii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 444    to 462, the second stretch of amino acid residues corresponds to one    of the amino acid sequences of SEQ ID NO's: 232 to 250 or of SEQ ID    NO's: 656 to 674; or (iii) when the first stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 656 to 674, the second stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 232    to 250 or of SEQ ID NO's: 444 to 462.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against ADAM8.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM8, that comprises threeor more stretches of amino acid residues, in which the first stretch ofamino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 232 to 250; the second stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 444 to 462; and the third stretch of amino acid residues is chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's:656 to 674.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against ADAM8.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 868 to 886. This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said amino acid sequence and one or more ofthe sequences of SEQ ID NO's: 868 to 886, in which the amino acidresidues that form the framework regions are disregarded. Also, suchamino acid sequences of the invention can be as further describedherein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAM8; and more in particularbind to ADAM8 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 232 to 250; and/or CDR2 is chosen from thegroup consisting of the amino acid sequences of SEQ ID NO's: 444 to 462;and/or CDR3 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 656 to 674.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 232 to 250; and CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 444 to 462; andCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 656 to 674.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAM8; and more in particularbind to ADAM8 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 868 to 886. This degree of aminoacid identity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said aminoacid sequence and one or more of the sequences of SEQ ID NO's: 868 to886, in which the amino acid residues that form the framework regionsare disregarded. Such amino acid sequences of the invention can be asfurther described herein.

In another more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM9 (such as a Nanobody ofthe invention, as further described herein), that comprises one or morestretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to    987;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004    to 1007;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024    to 1027;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to    987;-   b) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004    to 1007; and-   c) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024    to 1027;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against ADAM9.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM9, that comprises two ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to    987;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004    to 1007;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024    to 1027;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to    987;-   b) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004    to 1007; and-   c) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024    to 1027;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 251    to 271 and/or 984 to 987, the second stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 463    to 483 and/or 1004 to 1007 or of SEQ ID NO's: 675 to 695 and/or 1024    to 1027; (ii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 463    to 483 and/or 1004 to 1007, the second stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 251 to 271 and/or 984 to 987 or of SEQ ID NO's: 675 to 695    and/or 1024 to 1027; or (iii) when the first stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 675 to 695 and/or 1024 to 1027, the second stretch of amino    acid residues corresponds to one of the amino acid sequences of SEQ    ID NO's: 251 to 271 and/or 984 to 987 or of SEQ ID NO's: 463 to 483    and/or 1004 to 1007.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against ADAM9.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM9, that comprises threeor more stretches of amino acid residues, in which the first stretch ofamino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to    987;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004    to 1007;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024    to 1027;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 251 to 271 and/or 984 to 987; the second stretch ofamino acid residues is chosen from the group consisting of the aminoacid sequences of SEQ ID NO's: 463 to 483 and/or 1004 to 1007; and thethird stretch of amino acid residues is chosen from the group consistingof the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024 to1027.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against ADAM9.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 887 to 907 and 1044 to 1047. This degree of amino acid identitycan for example be determined by determining the degree of amino acididentity (in a manner described herein) between said amino acid sequenceand one or more of the sequences of SEQ ID NO's: 887 to 907 and 1044 to1047, in which the amino acid residues that form the framework regionsare disregarded. Also, such amino acid sequences of the invention can beas further described herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAM9; and more in particularbind to ADAM9 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to    987;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004    to 1007;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024    to 1027;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 251 to 271 and/or 984 to 987; and/or CDR2 ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 463 to 483 and/or 1004 to 1007; and/or CDR3 is chosen from thegroup consisting of the amino acid sequences of SEQ ID NO's: 675 to 695and/or 1024 to 1027.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to    987;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004    to 1007;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024    to 1027;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027; or any suitable fragment of such an amino    acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 251 to 271 and/or 984 to 987; and CDR2 ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 463 to 483 and/or 1004 to 1007; and CDR3 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 675 to 695 and/or1024 to 1027.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAM9; and more in particularbind to ADAM9 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 887 to 907 and 1044 to 1047. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and one or more of thesequences of SEQ ID NO's: 887 to 907 and 1044 to 1047, in which theamino acid residues that form the framework regions are disregarded.Such amino acid sequences of the invention can be as further describedherein.

In another even more specific, but non-limiting aspect, the inventionrelates to an amino acid sequence directed against ADAM10 (such as aNanobody of the invention, as further described herein), that comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;-   b) the amino acid sequences of SEQ ID NO's: 484 to 507; and-   c) the amino acid sequences of SEQ ID NO's: 696 to 719;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against ADAM10.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM10, that comprises two ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;-   b) the amino acid sequences of SEQ ID NO's: 484 to 507; and-   c) the amino acid sequences of SEQ ID NO's: 696 to 719;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 272    to 295, the second stretch of amino acid residues corresponds to one    of the amino acid sequences of SEQ ID NO's: 484 to 507 or of SEQ ID    NO's: 696 to 719; (ii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 484    to 507, the second stretch of amino acid residues corresponds to one    of the amino acid sequences of SEQ ID NO's: 272 to 295 or of SEQ ID    NO's: 696 to 719; or (iii) when the first stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 696 to 719, the second stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 272    to 295 or of SEQ ID NO's: 484 to 507.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against ADAM10.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM10, that comprises threeor more stretches of amino acid residues, in which the first stretch ofamino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 272 to 295; the second stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 484 to 507; and the third stretch of amino acid residues is chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's:696 to 719.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against ADAM10.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 908 to 931. This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said amino acid sequence and one or more ofthe sequences of SEQ ID NO's: 908 to 931, in which the amino acidresidues that form the framework regions are disregarded. Also, suchamino acid sequences of the invention can be as further describedherein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAM10; and more in particularbind to ADAM10 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 272 to 295; and/or CDR2 is chosen from thegroup consisting of the amino acid sequences of SEQ ID NO's: 484 to 507;and/or CDR3 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 696 to 719.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 272 to 295; and CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 484 to 507; andCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 696 to 719.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAM10; and more in particularbind to ADAM10 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 908 to 931. This degree of aminoacid identity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said aminoacid sequence and one or more of the sequences of SEQ ID NO's: 908 to931, in which the amino acid residues that form the framework regionsare disregarded. Such amino acid sequences of the invention can be asfurther described herein.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM17 (such as a Nanobody ofthe invention, as further described herein), that comprises one or morestretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to    992;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008    to 1012;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028    to 1032;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to    992;-   b) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008    to 1012; and-   c) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028    to 1032;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against ADAM17.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM17, that comprises two ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to    992;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008    to 1012;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028    to 1032;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to    992;-   b) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008    to 1012; and-   c) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028    to 1032;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 296    to 312 and/or 988 to 992, the second stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 508    to 524 and/or 1008 to 1012 or of SEQ ID NO's: 720 to 736 and/or 1028    to 1032; (ii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 508    to 524 and/or 1008 to 1012, the second stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 296 to 312 and/or 988 to 992 or of SEQ ID NO's: 720 to 736    and/or 1028 to 1032; or (iii) when the first stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 720 to 736 and/or 1028 to 1032, the second stretch of amino    acid residues corresponds to one of the amino acid sequences of SEQ    ID NO's: 296 to 312 and/or 988 to 992 or of SEQ ID NO's: 508 to 524    and/or 1008 to 1012.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against ADAM17.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAM17, that comprises threeor more stretches of amino acid residues, in which the first stretch ofamino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to    992;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008    to 1012;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028    to 1032;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 296 to 312 and/or 988 to 992; the second stretch ofamino acid residues is chosen from the group consisting of the aminoacid sequences of SEQ ID NO's: 508 to 524 and/or 1008 to 1012; and thethird stretch of amino acid residues is chosen from the group consistingof the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028 to1032.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against ADAM17.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 932 to 948 and 1048 to 1052 . This degree of amino acid identitycan for example be determined by determining the degree of amino acididentity (in a manner described herein) between said amino acid sequenceand one or more of the sequences of SEQ ID NO's: 932 to 948 and 1048 to1052, in which the amino acid residues that form the framework regionsare disregarded. Also, such amino acid sequences of the invention can beas further described herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAM17; and more in particularbind to ADAM17 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC50 value, asfurther described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to    992;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008    to 1012;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028    to 1032;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 296 to 312 and/or 988 to 992; and/or CDR2 ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 508 to 524 and/or 1008 to 1012; and/or CDR3 is chosen from thegroup consisting of the amino acid sequences of SEQ ID NO's: 720 to 736and/or 1028 to 1032.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to    992;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008    to 1012;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028    to 1032;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032; or any suitable fragment of such an amino    acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 296 to 312 and/or 988 to 992; and CDR2 ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 508 to 524 and/or 1008 to 1012; and CDR3 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 720 to 736 and/or1028 to 1032.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAM17; and more in particularbind to ADAM17 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 932 to 948 and 1048 to 1052. Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and one or more of thesequences of SEQ ID NO's: 932 to 948 and 1048 to 1052, in which theamino acid residues that form the framework regions are disregarded.Such amino acid sequences of the invention can be as further describedherein.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAMTS5 (such as a Nanobodyof the invention, as further described herein), that comprises one ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993;-   b) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; and-   c) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against ADAMTS5.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAMTS5, that comprises twoor more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993;-   b) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; and-   c) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 313    to 337 or 993, the second stretch of amino acid residues corresponds    to one of the amino acid sequences of SEQ ID NO's: 525 to 549 or    1013 or of SEQ ID NO's: 737 to 761 or 1033; (ii) when the first    stretch of amino acid residues corresponds to one of the amino acid    sequences of SEQ ID NO's: 525 to 549 or 1013, the second stretch of    amino acid residues corresponds to one of the amino acid sequences    of SEQ ID NO's: 313 to 337 or 993 or of SEQ ID NO's: 737 to 761 or    1033; or (iii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 737    to 761 or 1033, the second stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 313    to 337 or 993 or of SEQ ID NO's: 525 to 549 or 1013.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against ADAMTS5.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against ADAMTS5, that comprises threeor more stretches of amino acid residues, in which the first stretch ofamino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 313 to 337 or 993; the second stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 525 to 549 or 1013; and the third stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 737 to 761 or 1033.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against ADAMTS5.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 949 to 974 and 1053. This degree of amino acid identity can forexample be determined by determining the degree of amino acid identity(in a manner described herein) between said amino acid sequence and oneor more of the sequences of SEQ ID NO's: 949 to 974 and 1053, in whichthe amino acid residues that form the framework regions are disregarded.Also, such amino acid sequences of the invention can be as furtherdescribed herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAMTS5; and more in particularbind to ADAMTS5 with an affinity (suitably measured and/or expressed asa K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent),a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value,as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 313 to 337 or 993; and/or CDR2 is chosen fromthe group consisting of the amino acid sequences of SEQ ID NO's: 525 to549 or 1013; and/or CDR3 is chosen from the group consisting of theamino acid sequences of SEQ ID NO's: 737 to 761 or 1033.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 or 993;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 or 1013;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 or 1033; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 313 to 337 or 993; and CDR2 is chosen from thegroup consisting of the amino acid sequences of SEQ ID NO's: 525 to 549or 1013; and CDR3 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 737 to 761 or 1033.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to ADAMTS5; and more in particularbind to ADAMTS5 with an affinity (suitably measured and/or expressed asa K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent),a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value,as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 949 to 974 and 1053. This degree ofamino acid identity can for example be determined by determining thedegree of amino acid identity (in a manner described herein) betweensaid amino acid sequence and one or more of the sequences of SEQ IDNO's: 949 to 974 and 1053, in which the amino acid residues that formthe framework regions are disregarded. Such amino acid sequences of theinvention can be as further described herein.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanized) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequenceof the invention is a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody); is a single domain antibody (oran amino acid sequence that is suitable for use as a single domainantibody); is a “dAb” (or an amino acid sequence that is suitable foruse as a dAb); or is a Nanobody™ (including but not limited to V_(HH)sequence). Again, suitable framework sequences will be clear to theskilled person, for example on the basis the standard handbooks and thefurther disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acidsequences of the invention may contain one or more of Hallmark residues(as defined herein), such that the amino acid sequence of the inventionis a Nanobody™. Some preferred, but non-limiting examples of (suitablecombinations of) such framework sequences will become clear from thefurther disclosure herein.

Again, as generally described herein for the amino acid sequences of theinvention, it is also possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequenceas described herein (and in particular a CDR3 sequence), that is flankedon each side by (part of) a framework sequence (and in particular, partof the framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence). Such a fragment may also containa disulphide bridge, and in particular a disulphide bridge that linksthe two framework regions that precede and follow the CDR sequence,respectively (for the purpose of forming such a disulphide bridge,cysteine residues that naturally occur in said framework regions may beused, or alternatively cysteine residues may be synthetically added toor introduced into said framework regions). For a further description ofthese “Expedite fragments”, reference is again made to WO 03/050531, aswell as to as well as to the U.S. provisional application of Ablynx N.V.entitled “Peptides capable of binding to serum proteins” of Ablynx N.V.(inventors: Revets, Hilde Adi Pierrette; Kolkman, Joost Alexander; andHoogenboom, Hendricus Renerus Jacobus Mattheus) filed on Dec. 5, 2006(see also PCT/EP2007/063348).

In another aspect, the invention relates to a compound or construct, andin particular a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or moreamino acid sequences of the invention (or suitable fragments thereof),and optionally further comprises one or more other groups, residues,moieties or binding units. As will become clear to the skilled personfrom the further disclosure herein, such further groups, residues,moieties, binding units or amino acid sequences may or may not providefurther functionality to the amino acid sequence of the invention(and/or to the compound or construct in which it is present) and may ormay not modify the properties of the amino acid sequence of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound or construct is a (fusion) protein or (fusion) polypeptide. Ina preferred but non-limiting aspect, said one or more other groups,residues, moieties or binding units are immunoglobulin sequences. Evenmore preferably, said one or more other groups, residues, moieties orbinding units are chosen from the group consisting of domain antibodies,amino acid sequences that are suitable for use as a domain antibody,single domain antibodies, amino acid sequences that are suitable for useas a single domain antibody, “dAb's”, amino acid sequences that aresuitable for use as a dAb, or Nanobodies.

Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreamino acid sequences of the invention so as to provide a “derivative” ofan amino acid sequence or polypeptide of the invention, as furtherdescribed herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivatives as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more aminoacid sequences of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, moieties or binding units are amino acidsequences, the linkers may also be amino acid sequences, so that theresulting compound or construct is a fusion (protein) or fusion(polypeptide).

As will be clear from the further description above and herein, thismeans that the amino acid sequences of the invention can be used as“building blocks” to form polypeptides of the invention, i.e. bysuitably combining them with other groups, residues, moieties or bindingunits, in order to form compounds or constructs as described herein(such as, without limitations, the biparatopic. bi/multivalent andbi/multispecific polypeptides of the invention described herein) whichcombine within one molecule one or more desired properties or biologicalfunctions.

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more amino acid sequences of the invention to the one or morefurther groups, residues, moieties or binding units, optionally via theone or more suitable linkers, so as to provide the compound orpolypeptide of the invention. Polypeptides of the invention can also beprepared by a method which generally comprises at least the steps ofproviding a nucleic acid that encodes a polypeptide of the invention,expressing said nucleic acid in a suitable manner, and recovering theexpressed polypeptide of the invention. Such methods can be performed ina manner known per se, which will be clear to the skilled person, forexample on the basis of the methods and techniques further describedherein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedamino acid sequences form a further aspect of the invention.

For example, a multispecific polypeptide of the invention (as definedherein) may at least comprise at least one amino acid sequence (such asa Nanobody) against an ADAM proteinase and at least one binding unitagainst TNF (such as the Nanobodies described in the internationalapplications WO 06/122786 and WO 04/041862 of Ablynx N.V. or the dAb'sdescribed in WO 03/002609 or WO 04/003019). Such a multispecificpolypeptide may for example be used in the prevention and treatment ofinflammation and other diseases and disorders associated with TNF andthe TNF pathways. Reference is made to WO 06/122786, WO 04/041862, WO03/002609 or WO 04/003019 and WO 06/038027.

Another multispecific polypeptide of the invention may at least compriseat least one amino acid sequence (such as a Nanobody) against an ADAMproteinase (which amino acid sequence is preferably not inactivating)and at least one binding unit against a receptor so as to induceshedding and inactivation of said receptor. For example, such amultispecific polypeptide of the invention (as defined herein) may atleast comprise at least one amino acid sequence (such as a Nanobody)against an ADAM proteinase (which amino acid sequence is preferably notinactivating) and at least one binding unit against a TNF receptor (e.g.TNFR1 or TNFR2) so as to induce shedding and inactivation of said TNFreceptor (such as the dAb's described in WO 06/038027). Such amultispecific polypeptide may for example be used in the prevention andtreatment of inflammation and other diseases and disorders associatedwith TNF and the TNF pathways. Reference is again made to WO 06/122786,WO 04/041862, WO 03/002609 or WO 04/003019 and WO 06/038027.

Another multispecific polypeptide of the invention may at least compriseat least one amino acid sequence (such as a Nanobody) against an ADAMproteinase (which amino acid sequence is preferably not inactivating)and at least one binding unit against APP so as to cleave or solubilizeAPP aggregates.

Another multispecific polypeptide of the invention may at least compriseat least two amino acid sequence (such as two Nanobodies) againstdifferent ADAM proteinase so as to provide an increased and potentiallysynergistic modulating (and preferably inhibiting) effect on the ADAMproteinases.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise amino acid sequences or polypeptides ofthe invention that have been chemically modified to increase thehalf-life thereof (for example, by means of pegylation); amino acidsequences of the invention that comprise at least one additional bindingsite for binding to a serum protein (such as serum albumin); orpolypeptides of the invention that comprise at least one amino acidsequence of the invention that is linked to at least one moiety (and inparticular at least one amino acid sequence) that increases thehalf-life of the amino acid sequence of the invention. Examples ofpolypeptides of the invention that comprise such half-life extendingmoieties or amino acid sequences will become clear to the skilled personbased on the further disclosure herein; and for example include, withoutlimitation, polypeptides in which the one or more amino acid sequencesof the invention are suitable linked to one or more serum proteins orfragments thereof (such as (human) serum albumin or suitable fragmentsthereof) or to one or more binding units that can bind to serum proteins(such as, for example, domain antibodies, amino acid sequences that aresuitable for use as a domain antibody, single domain antibodies, aminoacid sequences that are suitable for use as a single domain antibody,“dAb's”, amino acid sequences that are suitable for use as a dAb, orNanobodies that can bind to serum proteins such as serum albumin (suchas human serum albumin), serum immunoglobulins such as IgG, ortransferrin; reference is made to the further description and referencesmentioned herein); polypeptides in which an amino acid sequence of theinvention is linked to an Fc portion (such as a human Fc) or a suitablepart or fragment thereof; or polypeptides in which the one or more aminoacid sequences of the invention are suitable linked to one or more smallproteins or peptides that can bind to serum proteins (such as, withoutlimitation, the proteins and peptides described in WO 91/01743, WO01/45746, WO 02/076489 and to the U.S. provisional application of AblynxN.V. entitled “Peptides capable of binding to serum proteins” of AblynxN.V. filed on Dec. 5, 2006 (see also PCT/EP2007/063348).

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodesan amino acid sequence of the invention or a polypeptide of theinvention (or a suitable fragment thereof). Such a nucleic acid willalso be referred to herein as a “nucleic acid of the invention” and mayfor example be in the form of a genetic construct, as further describedherein.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention and/or a polypeptideof the invention; and/or that contains a nucleic acid of the invention.Some preferred but non-limiting examples of such hosts or host cellswill become clear from the further description herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention, at leastone polypeptide of the invention (or a suitable fragment thereof) and/orat least one nucleic acid of the invention, and optionally one or morefurther components of such compositions known per se, i.e. depending onthe intended use of the composition. Such a product or composition mayfor example be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention further relates to methods for preparing or generating theamino acid sequences, polypeptides, nucleic acids, host cells, productsand compositions described herein. Some preferred but non-limitingexamples of such methods will become clear from the further descriptionherein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;    and-   b) screening said set, collection or library of amino acid sequences    for amino acid sequences that can bind to and/or have affinity for a    metalloproteinase from the ADAM family;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for a metalloproteinase from the ADAM family.

In such a method, the set, collection or library of amino acid sequencesmay be any suitable set, collection or library of amino acid sequences.For example, the set, collection or library of amino acid sequences maybe a set, collection or library of immunoglobulin sequences (asdescribed herein), such as a naïve set, collection or library ofimmunoglobulin sequences; a synthetic or semi-synthetic set, collectionor library of immunoglobulin sequences; and/or a set, collection orlibrary of immunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of amino acidsequences may be a set, collection or library of heavy chain variabledomains (such as V_(H) domains or V_(HH) domains) or of light chainvariable domains. For example, the set, collection or library of aminoacid sequences may be a set, collection or library of domain antibodiesor single domain antibodies, or may be a set, collection or library ofamino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofimmunoglobulin sequences, for example derived from a mammal that hasbeen suitably immunized with a metalloproteinase from the ADAM family orwith a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

In the above methods, the set, collection or library of amino acidsequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) amino acid sequences will beclear to the person skilled in the art, for example on the basis of thefurther disclosure herein. Reference is also made to the review byHoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating amino acid sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid    sequences;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for a metalloproteinase from the ADAM family;    and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulinsequence, the collection or sample of cells may for example be acollection or sample of B-cells. Also, in this method, the sample ofcells may be derived from a mammal that has been suitably immunized witha metalloproteinase from the ADAM family or with a suitable antigenicdeterminant based thereon or derived therefrom, such as an antigenicpart, fragment, region, domain, loop or other epitope thereof. In oneparticular aspect, said antigenic determinant may be an extracellularpart, region, domain, loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820 (2001).

In another aspect, the method for generating an amino acid sequencedirected against a metalloproteinase from the ADAM family may compriseat least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for a    metalloproteinase from the ADAM family;    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with a metalloproteinase from the ADAM family or witha suitable antigenic determinant based thereon or derived therefrom,such as an antigenic part, fragment, region, domain, loop or otherepitope thereof. In one particular aspect, said antigenic determinantmay be an extracellular part, region, domain, loop or otherextracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

The invention also relates to amino acid sequences that are obtained bythe above methods, or alternatively by a method that comprises the oneof the above methods and in addition at least the steps of determiningthe nucleotide sequence or amino acid sequence of said immunoglobulinsequence; and of expressing or synthesizing said amino acid sequence ina manner known per se, such as by expression in a suitable host cell orhost organism or by chemical synthesis.

Also, following the steps above, one or more amino acid sequences of theinvention may be suitably humanized (or alternatively camelized); and/orthe amino acid sequence(s) thus obtained may be linked to each other orto one or more other suitable amino acid sequences (optionally via oneor more suitable linkers) so as to provide a polypeptide of theinvention. Also, a nucleic acid sequence encoding an amino acid sequenceof the invention may be suitably humanized (or alternatively camelized)and suitably expressed; and/or one or more nucleic acid sequencesencoding an amino acid sequence of the invention may be linked to eachother or to one or more nucleic acid sequences that encode othersuitable amino acid sequences (optionally via nucleotide sequences thatencode one or more suitable linkers), after which the nucleotidesequence thus obtained may be suitably expressed so as to provide apolypeptide of the invention.

The invention further relates to applications and uses of the amino acidsequences, polypeptides, nucleic acids, host cells, products andcompositions described herein, as well as to methods for the preventionand/or treatment for diseases and disorders associated with ametalloproteinase from the ADAM family. Some preferred but non-limitingapplications and uses will become clear from the further descriptionherein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein, in which theinvention will be described and discussed in more detail with referenceto the Nanobodies of the invention and polypeptides of the inventioncomprising the same, which form some of the preferred aspects of theinvention.

As will become clear from the further description herein, Nanobodiesgenerally offer certain advantages (outlined herein) compared to “dAb's”or similar (single) domain antibodies or immunoglobulin sequences, whichadvantages are also provided by the Nanobodies of the invention.However, it will be clear to the skilled person that the more generalaspects of the teaching below can also be applied (either directly oranalogously) to other amino acid sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks, such as    Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd.Ed.),    Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et    al, eds., “Current protocols in molecular biology”, Green Publishing    and Wiley Interscience, New York (1987); Lewin, “Genes II”, John    Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of    Gene Manipulation: An Introduction to Genetic Engineering”, 2nd    edition, University of California Press, Berkeley, Calif. (1981);    Roitt et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh    (2001); Roitt et al., Roitt's Essential Immunology, 10^(th) Ed.    Blackwell Publishing, UK (2001); and Janeway et al., “Immunobiology”    (6th Ed.), Garland Science Publishing/Churchill Livingstone, N.Y.    (2005), as well as to the general background art cited herein;-   b) Unless indicated otherwise, the term “immunoglobulin sequence”    —whether used herein to refer to a heavy chain antibody or to a    conventional 4-chain antibody —is used as a general term to include    both the full-size antibody, the individual chains thereof, as well    as all parts, domains or fragments thereof (including but not    limited to antigen-binding domains or fragments such as V_(HH)    domains or V_(H)/V_(L) domains, respectively). In addition, the term    “sequence” as used herein (for example in terms like “immunoglobulin    sequence”, “antibody sequence”, “variable domain sequence”, “V_(HH)    sequence” or “protein sequence”), should generally be understood to    include both the relevant amino acid sequence as well as nucleic    acids or nucleotide sequences encoding the same, unless the context    requires a more limited interpretation. Also, the term “nucleotide    sequence” as used herein also encompasses a nucleic acid molecule    with said nucleotide sequence, so that the terms “nucleotide    sequence” and “nucleic acid” should be considered equivalent and are    used interchangeably herein;-   c) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   d) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code, as mentioned in Table    A-2;

TABLE A-2 one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0)Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0)Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimesalso considered to be a polar uncharged amino acid. ⁽²⁾Sometimes alsoconsidered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person.⁽⁴⁾As is known in the art, the charge of a His residue is greatlydependant upon even small shifts in pH, but a His residue can generallybe considered essentially uncharged at a pH of about 6.5.

-   e) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated by    dividing [the number of nucleotides in the first nucleotide sequence    that are identical to the nucleotides at the corresponding positions    in the second nucleotide sequence] by [the total number of    nucleotides in the first nucleotide sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of a nucleotide in the second nucleotide sequence—compared to the    first nucleotide sequence—is considered as a difference at a single    nucleotide (position).    -   Alternatively, the degree of sequence identity between two or        more nucleotide sequences may be calculated using a known        computer algorithm for sequence alignment such as NCBI Blast        v2.0, using standard settings.    -   Some other techniques, computer algorithms and settings for        determining the degree of sequence identity are for example        described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO        00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two nucleotide sequences in        accordance with the calculation method outlined hereinabove, the        nucleotide sequence with the greatest number of nucleotides will        be taken as the “first” nucleotide sequence, and the other        nucleotide sequence will be taken as the “second” nucleotide        sequence;-   f) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (also referred to herein    as “amino acid identity”) may be calculated by dividing [the number    of amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein.    -   Alternatively, the degree of sequence identity between two amino        acid sequences may be calculated using a known computer        algorithm, such as those mentioned above for determining the        degree of sequence identity for nucleotide sequences, again        using standard settings.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two amino acid sequences in        accordance with the calculation method outlined hereinabove, the        amino acid sequence with the greatest number of amino acid        residues will be taken as the “first” amino acid sequence, and        the other amino acid sequence will be taken as the “second”        amino acid sequence.    -   Also, in determining the degree of sequence identity between two        amino acid sequences, the skilled person may take into account        so-called “conservative” amino acid substitutions, which can        generally be described as amino acid substitutions in which an        amino acid residue is replaced with another amino acid residue        of similar chemical structure and which has little or        essentially no influence on the function, activity or other        biological properties of the polypeptide. Such conservative        amino acid substitutions are well known in the art, for example        from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and        WO 01/09300; and (preferred) types and/or combinations of such        substitutions may be selected on the basis of the pertinent        teachings from WO 04/037999 as well as WO 98/49185 and from the        further references cited therein.    -   Such conservative substitutions preferably are substitutions in        which one amino acid within the following groups (a)-(e) is        substituted by another amino acid residue within the same        group: (a) small aliphatic, nonpolar or slightly polar residues:        Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged        residues and their (uncharged) amides: Asp, Asn, Glu and        Gln; (c) polar, positively charged residues: His, Arg and        Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val        and Cys; and (e) aromatic residues: Phe, Tyr and Trp.    -   Particularly preferred conservative substitutions are as        follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or        into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into        Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile        into Leu or into Val; Leu into Ile or into Val; Lys into Arg,        into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe        into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp        into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into        Leu.    -   Any amino acid substitutions applied to the polypeptides        described herein may also be based on the analysis of the        frequencies of amino acid variations between homologous proteins        of different species developed by Schulz et al., Principles of        Protein Structure, Springer-Verlag, 1978, on the analyses of        structure forming potentials developed by Chou and Fasman,        Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978,        and on the analysis of hydrophobicity patterns in proteins        developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81:        140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132,        198 1, and Goldman et al., Ann Rev. Biophys. Chem. 15: 321-353,        1986, all incorporated herein in their entirety by reference.        Information on the primary, secondary and tertiary structure of        Nanobodies is given in the description herein and in the general        background art cited above. Also, for this purpose, the crystal        structure of a V_(HH) domain from a llama is for example given        by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803        (1996); Spinelli et al., Natural Structural Biology (1996); 3,        752-757; and Decanniere et al., Structure, Vol. 7, 4, 361        (1999). Further information about some of the amino acid        residues that in conventional V_(H) domains form the V_(H)/V_(L)        interface and potential camelizing substitutions on these        positions can be found in the prior art cited above.-   g) Amino acid sequences and nucleic acid sequences are said to be    “exactly the same” if they have 100% sequence identity (as defined    herein) over their entire length;-   h) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two amino    acid sequences can contain one, two or more such amino acid    differences;-   i) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this may mean that the latter    nucleotide sequence or amino acid sequence has been incorporated    into the first mentioned nucleotide sequence or amino acid sequence,    respectively, but more usually this generally means that the first    mentioned nucleotide sequence or amino acid sequence comprises    within its sequence a stretch of nucleotides or amino acid residues,    respectively, that has the same nucleotide sequence or amino acid    sequence, respectively, as the latter sequence, irrespective of how    the first mentioned sequence has actually been generated or obtained    (which may for example be by any suitable method described herein).    By means of a non-limiting example, when a Nanobody of the invention    is said to comprise a CDR sequence, this may mean that said CDR    sequence has been incorporated into the Nanobody of the invention,    but more usually this generally means that the Nanobody of the    invention contains within its sequence a stretch of amino acid    residues with the same amino acid sequence as said CDR sequence,    irrespective of how said Nanobody of the invention has been    generated or obtained. It should also be noted that when the latter    amino acid sequence has a specific biological or structural    function, it preferably has essentially the same, a similar or an    equivalent biological or structural function in the first mentioned    amino acid sequence (in other words, the first mentioned amino acid    sequence is preferably such that the latter sequence is capable of    performing essentially the same, a similar or an equivalent    biological or structural function). For example, when a Nanobody of    the invention is said to comprise a CDR sequence or framework    sequence, respectively, the CDR sequence and framework are    preferably capable, in said Nanobody, of functioning as a CDR    sequence or framework sequence, respectively. Also, when a    nucleotide sequence is said to comprise another nucleotide sequence,    the first mentioned nucleotide sequence is preferably such that,    when it is expressed into an expression product (e.g. a    polypeptide), the amino acid sequence encoded by the latter    nucleotide sequence forms part of said expression product (in other    words, that the latter nucleotide sequence is in the same reading    frame as the first mentioned, larger nucleotide sequence).-   j) A nucleic acid sequence or amino acid sequence is considered to    be “(in) essentially isolated (form)”—for example, compared to its    native biological source and/or the reaction medium or cultivation    medium from which it has been obtained—when it has been separated    from at least one other component with which it is usually    associated in said source or medium, such as another nucleic acid,    another protein/polypeptide, another biological component or    macromolecule or at least one contaminant, impurity or minor    component. In particular, a nucleic acid sequence or amino acid    sequence is considered “essentially isolated” when it has been    purified at least 2-fold, in particular at least 10-fold, more in    particular at least 100-fold, and up to 1000-fold or more. A nucleic    acid sequence or amino acid sequence that is “in essentially    isolated form” is preferably essentially homogeneous, as determined    using a suitable technique, such as a suitable chromatographical    technique, such as polyacrylamide-gel electrophoresis;-   k) The term “domain” as used herein generally refers to a globular    region of an amino acid sequence (such as an antibody chain, and in    particular to a globular region of a heavy chain antibody), or to a    polypeptide that essentially consists of such a globular region.    Usually, such a domain will comprise peptide loops (for example 3 or    4 peptide loops) stabilized, for example, as a sheet or by disulfide    bonds. The term “binding domain” refers to such a domain that is    directed against an antigenic determinant (as defined herein);-   l) The term “antigenic determinant” refers to the epitope on the    antigen recognized by the antigen-binding molecule (such as a    Nanobody or a polypeptide of the invention) and more in particular    by the antigen-binding site of said molecule. The terms “antigenic    determinant” and “epitope” may also be used interchangeably herein.-   m) An amino acid sequence (such as a Nanobody, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” said antigenic    determinant, epitope, antigen or protein.-   n) The term “specificity” refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as a    Nanobody or a polypeptide of the invention) molecule can bind. The    specificity of an antigen-binding protein can be determined based on    affinity and/or avidity. The affinity, represented by the    equilibrium constant for the dissociation of an antigen with an    antigen-binding protein (K_(D)), is a measure for the binding    strength between an antigenic determinant and an antigen-binding    site on the antigen-binding protein: the lesser the value of the    K_(D), the stronger the binding strength between an antigenic    determinant and the antigen-binding molecule (alternatively, the    affinity can also be expressed as the affinity constant (K_(A)),    which is 1/K_(D)). As will be clear to the skilled person (for    example on the basis of the further disclosure herein), affinity can    be determined in a manner known per se, depending on the specific    antigen of interest. Avidity is the measure of the strength of    binding between an antigen-binding molecule (such as a Nanobody or    polypeptide of the invention) and the pertinent antigen. Avidity is    related to both the affinity between an antigenic determinant and    its antigen binding site on the antigen-binding molecule and the    number of pertinent binding sites present on the antigen-binding    molecule. Typically, antigen-binding proteins (such as the amino    acid sequences, Nanobodies and/or polypeptides of the invention)    will bind to their antigen with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter    (i.e. with an association constant (K_(A)) of 10⁵ to 10¹²    liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more    and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value    greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)    liters/mol is generally considered to indicate non-specific binding.    Preferably, a monovalent immunoglobulin sequence of the invention    will bind to the desired antigen with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM. Specific binding of an antigen-binding protein    to an antigen or antigenic determinant can be determined in any    suitable manner known per se, including, for example, Scatchard    analysis and/or competitive binding assays, such as    radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich    competition assays, and the different variants thereof known per se    in the art; as well as the other techniques mentioned herein.    -   The dissociation constant may be the actual or apparent        dissociation constant, as will be clear to the skilled person.        Methods for determining the dissociation constant will be clear        to the skilled person, and for example include the techniques        mentioned herein. In this respect, it will also be clear that it        may not be possible to measure dissociation constants of more        then 10⁻⁴ moles/liter or 10⁻³ moles/liter (e.g., of 10⁻²        moles/liter). Optionally, as will also be clear to the skilled        person, the (actual or apparent) dissociation constant may be        calculated on the basis of the (actual or apparent) association        constant (K_(A)), by means of the relationship [K_(D)=1/K_(A)].    -   The affinity denotes the strength or stability of a molecular        interaction. The affinity is commonly given as by the K_(D), or        dissociation constant, which has units of mol/liter (or M). The        affinity can also be expressed as an association constant,        K_(A), which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or        M⁻¹). In the present specification, the stability of the        interaction between two molecules (such as an amino acid        sequence, Nanobody or polypeptide of the invention and its        intended target) will mainly be expressed in terms of the K_(D)        value of their interaction; it being clear to the skilled person        that in view of the relation K_(A)=1/K_(D), specifying the        strength of molecular interaction by its K_(D) value can also be        used to calculate the corresponding K_(A) value. The K_(D)-value        characterizes the strength of a molecular interaction also in a        thermodynamic sense as it is related to the free energy (DG) of        binding by the well known relation DG=RT.ln(K_(D)) (equivalently        DG=−RT.ln(K_(A))), where R equals the gas constant, T equals the        absolute temperature and In denotes the natural logarithm.    -   The K_(D) for biological interactions which are considered        meaningful (e.g. specific) are typically in the range of 10⁻¹⁰M        (0.1 nM) to 10⁻⁵M (10000 nM). The stronger an interaction is,        the lower is its K_(D).    -   The K_(D) can also be expressed as the ratio of the dissociation        rate constant of a complex, denoted as k_(off), to the rate of        its association, denoted k_(on) (so that K_(D)=k_(off)/k_(on)        and K_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹        (where s is the SI unit notation of second). The on-rate k_(on)        has units M⁻¹s⁻¹. The on-rate may vary between 10² M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, approaching the diffusion-limited association        rate constant for bimolecular interactions. The off-rate is        related to the half-life of a given molecular interaction by the        relation t_(1/2)=ln(2)/k_(off). The off-rate may vary between        10⁻⁶ s⁻¹ (near irreversible complex with a t_(1/2) of multiple        days) to 1 s⁻¹ (t_(1/2)=0.69 s).    -   The affinity of a molecular interaction between two molecules        can be measured via different techniques known per se, such as        the well known surface plasmon resonance (SPR) biosensor        technique (see for example Ober et al., Intern. Immunology, 13,        1551-1559, 2001) where one molecule is immobilized on the        biosensor chip and the other molecule is passed over the        immobilized molecule under flow conditions yielding k_(on),        k_(off) measurements and hence K_(D) (or K_(A)) values. This can        for example be performed using the well-known BIACORE        instruments.    -   It will also be clear to the skilled person that the measured        K_(D) may correspond to the apparent K_(D) if the measuring        process somehow influences the intrinsic binding affinity of the        implied molecules for example by artefacts related to the        coating on the biosensor of one molecule. Also, an apparent        K_(D) may be measured if one molecule contains more than one        recognition sites for the other molecule. In such situation the        measured affinity may be affected by the avidity of the        interaction by the two molecules.    -   Another approach that may be used to assess affinity is the        2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of        Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This        method establishes a solution phase binding equilibrium        measurement and avoids possible artefacts relating to adsorption        of one of the molecules on a support such as plastic.    -   However, the accurate measurement of K_(D) may be quite        labor-intensive and as consequence, often apparent K_(D) values        are determined to assess the binding strength of two molecules.        It should be noted that as long all measurements are made in a        consistent way (e.g. keeping the assay conditions unchanged)        apparent K_(D) measurements can be used as an approximation of        the true K_(D) and hence in the present document K_(D) and        apparent K_(D) should be treated with equal importance or        relevance. Finally, it should be noted that in many situations        the experienced scientist may judge it to be convenient to        determine the binding affinity relative to some reference        molecule. For example, to assess the binding strength between        molecules A and B, one may e.g. use a reference molecule C that        is known to bind to B and that is suitably labelled with a        fluorophore or chromophore group or other chemical moiety, such        as biotin for easy detection in an ELISA or FACS (Fluorescent        activated cell sorting) or other format (the fluorophore for        fluorescence detection, the chromophore for light absorption        detection, the biotin for streptavidin-mediated ELISA        detection). Typically, the reference molecule C is kept at a        fixed concentration and the concentration of A is varied for a        given concentration or amount of B. As a result an IC₅₀ value is        obtained corresponding to the concentration of A at which the        signal measured for C in absence of A is halved. Provided        K_(D ref), the K_(D) of the reference molecule, is known, as        well as the total concentration c_(ref) of the reference        molecule, the apparent K_(D) for the interaction A-B can be        obtained from following formula:        K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if        c_(ref)<<K_(D ref), K_(D)≈IC₅₀. Provided the measurement of the        IC₅₀ is performed in a consistent way (e.g. keeping c_(ref)        fixed) for the binders that are compared, the strength or        stability of a molecular interaction can be assessed by the IC₅₀        and this measurement is judged as equivalent to K_(D) or to        apparent K_(D) throughout this text.-   o) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as the time taken for the    serum concentration of the amino acid sequence, compound or    polypeptide to be reduced by 50%, in vivo, for example due to    degradation of the sequence or compound and/or clearance or    sequestration of the sequence or compound by natural mechanisms. The    in vivo half-life of an amino acid sequence, compound or polypeptide    of the invention can be determined in any manner known per se, such    as by pharmacokinetic analysis. Suitable techniques will be clear to    the person skilled in the art, and may for example generally involve    the steps of suitably administering to a warm-blooded animal (i.e.    to a human or to another suitable mammal, such as a mouse, rabbit,    rat, pig, dog or a primate, for example monkeys from the genus    Macaca (such as, and in particular, cynomolgus monkeys (Macaca    fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon    (Papio ursinus)) a suitable dose of the amino acid sequence,    compound or polypeptide of the invention; collecting blood samples    or other samples from said animal; determining the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention in said blood sample; and calculating, from (a plot    of) the data thus obtained, the time until the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention has been reduced by 50% compared to the initial level    upon dosing. Reference is for example made to the Experimental Part    below, as well as to the standard handbooks, such as Kenneth, A et    al: Chemical Stability of Pharmaceuticals: A Handbook for    Pharmacists and Peters et al, Pharmacokinete analysis: A Practical    Approach (1996). Reference is also made to “Pharmacokinetics”, M    Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition    (1982).    -   As will also be clear to the skilled person (see for example        pages 6 and 7 of WO 04/003019 and in the further references        cited therein), the half-life can be expressed using parameters        such as the t1/2-alpha, t1/2-beta and the area under the curve        (AUC). In the present specification, an “increase in half-life”        refers to an increase in any one of these parameters, such as        any two of these parameters, or essentially all three these        parameters. As used herein “increase in half-life” or “increased        half-life” in particular refers to an increase in the t1/2-beta,        either with or without an increase in the t1/2-alpha and/or the        AUC or both.-   p) In the context of the present invention, “modulating” or “to    modulate” generally means either reducing or inhibiting the activity    of, or alternatively increasing the activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay. In particular, “modulating” or “to modulate” may mean either    reducing or inhibiting the activity of, or alternatively increasing    a (relevant or intended) biological activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay (which will usually depend on the target or antigen involved),    by at least 1%, preferably at least 5%, such as at least 10% or at    least 25%, for example by at least 50%, at least 60%, at least 70%,    at least 80%, or 90% or more, compared to activity of the target or    antigen in the same assay under the same conditions but without the    presence of the construct of the invention.    -   As will be clear to the skilled person, “modulating” may also        involve effecting a change (which may either be an increase or a        decrease) in affinity, avidity, specificity and/or selectivity        of a target or antigen for one or more of its ligands, binding        partners, partners for association into a homomultimeric or        heteromultimeric form, or substrates; and/or effecting a change        (which may either be an increase or a decrease) in the        sensitivity of the target or antigen for one or more conditions        in the medium or surroundings in which the target or antigen is        present (such as pH, ion strength, the presence of co-factors,        etc.), compared to the same conditions but without the presence        of the construct of the invention. As will be clear to the        skilled person, this may again be determined in any suitable        manner and/or using any suitable assay known per se, depending        on the target or antigen involved.    -   “Modulating” may also mean effecting a change (i.e. an activity        as an agonist, as an antagonist or as a reverse agonist,        respectively, depending on the target or antigen and the desired        biological or physiological effect) with respect to one or more        biological or physiological mechanisms, effects, responses,        functions, pathways or activities in which the target or antigen        (or in which its substrate(s), ligand(s) or pathway(s) are        involved, such as its signalling pathway or metabolic pathway        and their associated biological or physiological effects) is        involved. Again, as will be clear to the skilled person, such an        action as an agonist or an antagonist may be determined in any        suitable manner and/or using any suitable (in vitro and usually        cellular or in assay) assay known per se, depending on the        target or antigen involved. In particular, an action as an        agonist or antagonist may be such that an intended biological or        physiological activity is increased or decreased, respectively,        by at least 1%, preferably at least 5%, such as at least 10% or        at least 25%, for example by at least 50%, at least 60%, at        least 70%, at least 80%, or 90% or more, compared to the        biological or physiological activity in the same assay under the        same conditions but without the presence of the construct of the        invention.    -   Modulating may for example also involve allosteric modulation of        the target or antigen; and/or reducing or inhibiting the binding        of the target or antigen to one of its substrates or ligands        and/or competing with a natural ligand, substrate for binding to        the target or antigen. Modulating may also involve activating        the target or antigen or the mechanism or pathway in which it is        involved. Modulating may for example also involve effecting a        change in respect of the folding or confirmation of the target        or antigen, or in respect of the ability of the target or        antigen to fold, to change its confirmation (for example, upon        binding of a ligand), to associate with other (sub)units, or to        disassociate. Modulating may for example also involve effecting        a change in the ability of the target or antigen to transport        other compounds or to serve as a channel for other compounds        (such as ions).    -   Modulating may be reversible or irreversible, but for        pharmaceutical and pharmacological purposes will usually be in a        reversible manner.-   q) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein).-   r) An amino acid sequence or polypeptide is said to be “specific    for” a first target or antigen compared to a second target or    antigen when is binds to the first antigen with an affinity (as    described above, and suitably expressed as a K_(D) value, K_(A)    value, K_(off) rate and/or K_(on) rate) that is at least 10 times,    such as at least 100 times, and preferably at least 1000 times, and    up to 10.000 times or more better than the affinity with which said    amino acid sequence or polypeptide binds to the second target or    polypeptide. For example, the first antigen may bind to the target    or antigen with a K_(D) value that is at least 10 times less, such    as at least 100 times less, and preferably at least 1000 times less,    such as 10.000 times less or even less than that, than the K_(D)    with which said amino acid sequence or polypeptide binds to the    second target or polypeptide.    -   Preferably, when an amino acid sequence or polypeptide is        “specific for” a first target or antigen compared to a second        target or antigen, it is directed against (as defined herein)        said first target or antigen, but not directed against said        second target or antigen.-   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an amino acid    sequence or other binding agents (such as a polypeptide of the    invention) to interfere with the binding of other amino acid    sequences or binding agents of the invention to a given target. The    extend to which an amino acid sequence or other binding agents of    the invention is able to interfere with the binding of another to    [target], and therefore whether it can be said to cross-block    according to the invention, can be determined using competition    binding assays. One particularly suitable quantitative assay uses a    Biacore machine which can measure the extent of interactions using    surface plasmon resonance technology. Another suitable quantitative    cross-blocking assay uses an ELISA-based approach to measure    competition between amino acid sequence or another binding agents in    terms of their binding to the target.    -   The following generally describes a suitable Biacore assay for        determining whether an amino acid sequence or other binding        agent cross-blocks or is capable of cross-blocking according to        the invention. It will be appreciated that the assay can be used        with any of the amino acid sequence or other binding agents        described herein. The Biacore machine (for example the        Biacore 3000) is operated in line with the manufacturer's        recommendations. Thus in one cross-blocking assay, the target        protein is coupled to a CM5 Biacore chip using standard amine        coupling chemistry to generate a surface that is coated with the        target. Typically 200-800 resonance units of the target would be        coupled to the chip (an amount that gives easily measurable        levels of binding but that is readily saturable by the        concentrations of test reagent being used). Two test amino acid        sequences (termed A* and B*) to be assessed for their ability to        cross-block each other are mixed at a one to one molar ratio of        binding sites in a suitable buffer to create the test mixture.        When calculating the concentrations on a binding site basis the        molecular weight of an amino acid sequence is assumed to be the        total molecular weight of the amino acid sequence divided by the        number of target binding sites on that amino acid sequence. The        concentration of each amino acid sequence in the test mix should        be high enough to readily saturate the binding sites for that        amino acid sequence on the target molecules captured on the        Biacore chip. The amino acid sequences in the mixture are at the        same molar concentration (on a binding basis) and that        concentration would typically be between 1.00 and 1.5 micromolar        (on a binding site basis). Separate solutions containing A*        alone and B* alone are also prepared. A* and B* in these        solutions should be in the same buffer and at the same        concentration as in the test mix. The test mixture is passed        over the target-coated Biacore chip and the total amount of        binding recorded. The chip is then treated in such a way as to        remove the bound amino acid sequences without damaging the        chip-bound target. Typically this is done by treating the chip        with 30 mM HCl for 60 seconds. The solution of A* alone is then        passed over the target-coated surface and the amount of binding        recorded. The chip is again treated to remove all of the bound        amino acid sequences without damaging the chip-bound target. The        solution of B* alone is then passed over the target-coated        surface and the amount of binding recorded. The maximum        theoretical binding of the mixture of A* and B* is next        calculated, and is the sum of the binding of each amino acid        sequence when passed over the target surface alone. If the        actual recorded binding of the mixture is less than this        theoretical maximum then the two amino acid sequences are        cross-blocking each other. Thus, in general, a cross-blocking        amino acid sequence or other binding agent according to the        invention is one which will bind to the target in the above        Biacore cross-blocking assay such that during the assay and in        the presence of a second amino acid sequence or other binding        agent of the invention the recorded binding is between 80% and        0.1% (e.g. 80% to 4%) of the maximum theoretical binding,        specifically between 75% and 0.1% (e.g. 75% to 4%) of the        maximum theoretical binding, and more specifically between 70%        and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as        just defined above) of the two amino acid sequences or binding        agents in combination. The Biacore assay described above is a        primary assay used to determine if amino acid sequences or other        binding agents cross-block each other according to the        invention. On rare occasions particular amino acid sequences or        other binding agents may not bind to target coupled via amine        chemistry to a CM5 Biacore chip (this usually occurs when the        relevant binding site on target is masked or destroyed by the        coupling to the chip). In such cases cross-blocking can be        determined using a tagged version of the target, for example a        N-terminal His-tagged version (R & D Systems, Minneapolis,        Minn., USA; 2005 cat #1406-ST-025). In this particular format,        an anti-His amino acid sequence would be coupled to the Biacore        chip and then the His-tagged target would be passed over the        surface of the chip and captured by the anti-His amino acid        sequence. The cross blocking analysis would be carried out        essentially as described above, except that after each chip        regeneration cycle, new His-tagged target would be loaded back        onto the anti-His amino acid sequence coated surface. In        addition to the example given using N-terminal His-tagged        [target], C-terminal His-tagged target could alternatively be        used. Furthermore, various other tags and tag binding protein        combinations that are known in the art could be used for such a        cross-blocking analysis (e.g. HA tag with anti-HA antibodies;        FLAG tag with anti-FLAG antibodies; biotin tag with        streptavidin).    -   The following generally describes an ELISA assay for determining        whether an amino acid sequence or other binding agent directed        against a target cross-blocks or is capable of cross-blocking as        defined herein. It will be appreciated that the assay can be        used with any of the amino acid sequences (or other binding        agents such as polypeptides of the invention) described herein.        The general principal of the assay is to have an amino acid        sequence or binding agent that is directed against the target        coated onto the wells of an ELISA plate. An excess amount of a        second, potentially cross-blocking, anti-target amino acid        sequence is added in solution (i.e. not bound to the ELISA        plate). A limited amount of the target is then added to the        wells. The coated amino acid sequence and the amino acid        sequence in solution compete for binding of the limited number        of target molecules. The plate is washed to remove excess target        that has not been bound by the coated amino acid sequence and to        also remove the second, solution phase amino acid sequence as        well as any complexes formed between the second, solution phase        amino acid sequence and target. The amount of bound target is        then measured using a reagent that is appropriate to detect the        target. An amino acid sequence in solution that is able to        cross-block the coated amino acid sequence will be able to cause        a decrease in the number of target molecules that the coated        amino acid sequence can bind relative to the number of target        molecules that the coated amino acid sequence can bind in the        absence of the second, solution phase, amino acid sequence. In        the instance where the first amino acid sequence, e.g. an Ab-X,        is chosen to be the immobilized amino acid sequence, it is        coated onto the wells of the ELISA plate, after which the plates        are blocked with a suitable blocking solution to minimize        non-specific binding of reagents that are subsequently added. An        excess amount of the second amino acid sequence, i.e. Ab-Y, is        then added to the ELISA plate such that the moles of Ab-Y        [target] binding sites per well are at least 10 fold higher than        the moles of Ab-X [target] binding sites that were used, per        well, during the coating of the ELISA plate. [target] is then        added such that the moles of [target] added per well are at        least 25-fold lower than the moles of Ab-X [target] binding        sites that were used for coating each well. Following a suitable        incubation period the ELISA plate is washed and a reagent for        detecting the target is added to measure the amount of target        specifically bound by the coated anti-[target] amino acid        sequence (in this case Ab-X). The background signal for the        assay is defined as the signal obtained in wells with the coated        amino acid sequence (in this case Ab-X), second solution phase        amino acid sequence (in this case Ab-Y), [target] buffer only        (i.e. no target) and target detection reagents. The positive        control signal for the assay is defined as the signal obtained        in wells with the coated amino acid sequence (in this case        Ab-X), second solution phase amino acid sequence buffer only        (i.e. no second solution phase amino acid sequence), target and        target detection reagents. The ELISA assay may be run in such a        manner so as to have the positive control signal be at least 6        times the background signal. To avoid any artefacts (e.g.        significantly different affinities between Ab-X and Ab-Y for        [target]) resulting from the choice of which amino acid sequence        to use as the coating amino acid sequence and which to use as        the second (competitor) amino acid sequence, the cross-blocking        assay may to be run in two formats: 1) format 1 is where Ab-X is        the amino acid sequence that is coated onto the ELISA plate and        Ab-Y is the competitor amino acid sequence that is in solution        and 2) format 2 is where Ab-Y is the amino acid sequence that is        coated onto the ELISA plate and Ab-X is the competitor amino        acid sequence that is in solution. Ab-X and Ab-Y are defined as        cross-blocking if, either in format 1 or in format 2, the        solution phase anti-target amino acid sequence is able to cause        a reduction of between 60% and 100%, specifically between 70%        and 100%, and more specifically between 80% and 100%, of the        target detection signal {i.e. the amount of target bound by the        coated amino acid sequence) as compared to the target detection        signal obtained in the absence of the solution phase anti-target        amino acid sequence (i.e. the positive control wells).-   t) As further described herein, the total number of amino acid    residues in a Nanobody can be in the region of 110-120, is    preferably 112-115, and is most preferably 113. It should however be    noted that parts, fragments, analogs or derivatives (as further    described herein) of a Nanobody are not particularly limited as to    their length and/or size, as long as such parts, fragments, analogs    or derivatives meet the further requirements outlined herein and are    also preferably suitable for the purposes described herein;-   u) The amino acid residues of a Nanobody are numbered according to    the general numbering for V_(H) domains given by Kabat et al.    (“Sequence of proteins of immunological interest”, US Public Health    Services, NIH Bethesda, Md., Publication No. 91), as applied to    V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, J. Immunol. Methods Jun. 23, 2000; 240 (1-2): 185-195    (see for example FIG. 2 of this publication); or referred to herein.    According to this numbering, FR1 of a Nanobody comprises the amino    acid residues at positions 1-30, CDR1 of a Nanobody comprises the    amino acid residues at positions 31-35, FR2 of a Nanobody comprises    the amino acids at positions 36-49, CDR2 of a Nanobody comprises the    amino acid residues at positions 50-65, FR3 of a Nanobody comprises    the amino acid residues at positions 66-94, CDR3 of a Nanobody    comprises the amino acid residues at positions 95-102, and FR4 of a    Nanobody comprises the amino acid residues at positions 103-113. [In    this respect, it should be noted that—as is well known in the art    for V_(H) domains and for V_(HH) domains—the total number of amino    acid residues in each of the CDR's may vary and may not correspond    to the total number of amino acid residues indicated by the Kabat    numbering (that is, one or more positions according to the Kabat    numbering may not be occupied in the actual sequence, or the actual    sequence may contain more amino acid residues than the number    allowed for by the Kabat numbering). This means that, generally, the    numbering according to Kabat may or may not correspond to the actual    numbering of the amino acid residues in the actual sequence.    Generally, however, it can be said that, according to the numbering    of Kabat and irrespective of the number of amino acid residues in    the CDR's, position 1 according to the Kabat numbering corresponds    to the start of FR1 and vice versa, position 36 according to the    Kabat numbering corresponds to the start of FR2 and vice versa,    position 66 according to the Kabat numbering corresponds to the    start of FR3 and vice versa, and position 103 according to the Kabat    numbering corresponds to the start of FR4 and vice versa.].    -   Alternative methods for numbering the amino acid residues of        V_(H) domains, which methods can also be applied in an analogous        manner to V_(HH) domains from Camelids and to Nanobodies, are        the method described by Chothia et al. (Nature 342, 877-883        (1989)), the so-called “AbM definition” and the so-called        “contact definition”. However, in the present description,        claims and figures, the numbering according to Kabat as applied        to V_(HH) domains by Riechmann and Muyldermans will be followed,        unless indicated otherwise; and-   v) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, to the review article by Muyldermans in Reviews in MolecularBiotechnology 74(2001), 277-302; as well as to the following patentapplications, which are mentioned as general background art: WO94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel;WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N.V. and Ablynx N.V.; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 (=EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the furtherpublished patent applications by Ablynx N.V. Reference is also made tothe further prior art mentioned in these applications, and in particularto the list of references mentioned on pages 41-43 of the Internationalapplication WO 06/040153, which list and references are incorporatedherein by reference.

In accordance with the terminology used in the art (see the abovereferences), the variable domains present in naturally occurring heavychain antibodies will also be referred to as “V_(HH) domains”, in orderto distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies (which will be referred tohereinbelow as “V_(H) domains”) and from the light chain variabledomains that are present in conventional 4-chain antibodies (which willbe referred to hereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have anumber of unique structural characteristics and functional propertieswhich make isolated V_(HH) domains (as well as Nanobodies based thereon,which share these structural characteristics and functional propertieswith the naturally occurring V_(HH) domains) and proteins containing thesame highly advantageous for use as functional antigen-binding domainsor proteins. In particular, and without being limited thereto, V_(HH)domains (which have been “designed” by nature to functionally bind to anantigen without the presence of, and without any interaction with, alight chain variable domain) and Nanobodies can function as a single,relatively small, functional antigen-binding structural unit, domain orprotein. This distinguishes the V_(HH) domains from the V_(H) and V_(L)domains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or domains, but need to be combined in some form or another toprovide a functional antigen-binding unit (as in for exampleconventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains andNanobodies as single antigen-binding proteins or as antigen bindingdomains (i.e. as part of a larger protein or polypeptide) offers anumber of significant advantages over the use of conventional V_(H) andV_(L) domains, scFv's or conventional antibody fragments (such as Fab-or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spatial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   V_(HH) domains and Nanobodies can be expressed from a single        gene and require no post-translational folding or modifications;    -   V_(HH) domains and Nanobodies can easily be engineered into        multivalent and multispecific formats (as further discussed        herein);    -   V_(HH) domains and Nanobodies are highly soluble and do not have        a tendency to aggregate (as with the mouse-derived “dAb's”        described by Ward et al., Nature, Vol. 341, 1989, p. 544);    -   V_(HH) domains and Nanobodies are highly stable to heat, pH,        proteases and other denaturing agents or conditions (see for        example Ewert et al, supra);    -   V_(HH) domains and Nanobodies are easy and relatively cheap to        prepare, even on a scale required for production. For example,        V_(HH) domains, Nanobodies and proteins/polypeptides containing        the same can be produced using microbial fermentation (e.g. as        further described below) and do not require the use of mammalian        expression systems, as with for example conventional antibody        fragments;    -   V_(HH) domains and Nanobodies are relatively small        (approximately 15 kDa, or 10 times smaller than a conventional        IgG) compared to conventional 4-chain antibodies and        antigen-binding fragments thereof, and therefore show high(er)        penetration into tissues (including but not limited to solid        tumors and other dense tissues) than such conventional 4-chain        antibodies and antigen-binding fragments thereof;    -   V_(HH) domains and Nanobodies can show so-called cavity-binding        properties (inter alia due to their extended CDR3 loop, compared        to conventional V_(H) domains) and can therefore also access        targets and epitopes not accessible to conventional 4-chain        antibodies and antigen-binding fragments thereof. For example,        it has been shown that V_(HH) domains and Nanobodies can inhibit        enzymes (see for example WO 97/49805; Transue et al., Proteins        Sep. 1, 1998; 32(4): 515-22; Lauwereys et al., EMBO J. Jul. 1,        1998; 17(13): 3512-20).

In a specific and preferred aspect, the invention provides Nanobodiesagainst a metalloproteinase from the ADAM family, and in particularNanobodies against a metalloproteinase from the ADAM family from awarm-blooded animal, and more in particular Nanobodies against ametalloproteinase from the ADAM family from a mammal, and especiallyNanobodies against human a metalloproteinase from the ADAM family; aswell as proteins and/or polypeptides comprising at least one suchNanobody.

In particular, the invention provides Nanobodies against ametalloproteinase from the ADAM family, and proteins and/or polypeptidescomprising the same, that have improved therapeutic and/orpharmacological properties and/or other advantageous properties (suchas, for example, improved ease of preparation and/or reduced costs ofgoods), compared to conventional antibodies against a metalloproteinasefrom the ADAM family or fragments thereof, compared to constructs thatcould be based on such conventional antibodies or antibody fragments(such as Fab′ fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies”and other multispecific constructs (see for example the review byHolliger and Hudson, Nat Biotechnol. September 2005; 23(9):1126-36)),and also compared to the so-called “dAb's” or similar (single) domainantibodies that may be derived from variable domains of conventionalantibodies. These improved and advantageous properties will become clearfrom the further description herein, and for example include, withoutlimitation, one or more of:

-   -   increased affinity and/or avidity for a metalloproteinase from        the ADAM family, either in a monovalent format, in a multivalent        format (for example in a bivalent format) and/or in a        multispecific format (for example one of the multispecific        formats described hereinbelow);    -   better suitability for formatting in a multivalent format (for        example in a bivalent format);    -   better suitability for formatting in a multispecific format (for        example one of the multispecific formats described hereinbelow);    -   improved suitability or susceptibility for “humanizing”        substitutions (as defined herein);    -   less immunogenicity, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased stability, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased specificity towards a metalloproteinase from the ADAM        family, either in a monovalent format, in a multivalent format        (for example in a bivalent format) and/or in a multispecific        format (for example one of the multispecific formats described        hereinbelow);    -   decreased or where desired increased cross-reactivity with a        metalloproteinase from the ADAM family from different species;        and/or    -   one or more other improved properties desirable for        pharmaceutical use (including prophylactic use and/or        therapeutic use) and/or for diagnostic use (including but not        limited to use for imaging purposes), either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described hereinbelow).

As generally described herein for the amino acid sequences of theinvention, the Nanobodies of the invention are preferably in essentiallyisolated form (as defined herein), or form part of a protein orpolypeptide of the invention (as defined herein), which may comprise oressentially consist of one or more Nanobodies of the invention and whichmay optionally further comprise one or more further amino acid sequences(all optionally linked via one or more suitable linkers). For example,and without limitation, the one or more amino acid sequences of theinvention may be used as a binding unit in such a protein orpolypeptide, which may optionally contain one or more further amino acidsequences that can serve as a binding unit (i.e. against one or moreother targets than a metalloproteinase from the ADAM family), so as toprovide a monovalent, multivalent or multispecific polypeptide of theinvention, respectively, all as described herein. In particular, such aprotein or polypeptide may comprise or essentially consist of one ormore Nanobodies of the invention and optionally one or more (other)Nanobodies (i.e. directed against other targets than a metalloproteinasefrom the ADAM family), all optionally linked via one or more suitablelinkers, so as to provide a monovalent, multivalent or multispecificNanobody construct, respectively, as further described herein. Suchproteins or polypeptides may also be in essentially isolated form (asdefined herein).

In a Nanobody of the invention, the binding site for binding against ametalloproteinase from the ADAM family is preferably formed by the CDRsequences. Optionally, a Nanobody of the invention may also, and inaddition to the at least one binding site for binding against ametalloproteinase from the ADAM family, contain one or more furtherbinding sites for binding against other antigens, proteins or targets.For methods and positions for introducing such second binding sites,reference is for example made to Keck and Huston, Biophysical Journal,71, October 1996, 2002-2011; EP 0 640 130 and WO 06/07260.

As generally described herein for the amino acid sequences of theinvention, when a Nanobody of the invention (or a polypeptide of theinvention comprising the same) is intended for administration to asubject (for example for therapeutic and/or diagnostic purposes asdescribed herein), it is preferably directed against human ametalloproteinase from the ADAM family; whereas for veterinary purposes,it is preferably directed against a metalloproteinase from the ADAMfamily from the species to be treated. Also, as with the amino acidsequences of the invention, a Nanobody of the invention may or may notbe cross-reactive (i.e. directed against a metalloproteinase from theADAM family from two or more species of mammal, such as against human ametalloproteinase from the ADAM family and a metalloproteinase from theADAM family from at least one of the species of mammal mentionedherein).

Also, again as generally described herein for the amino acid sequencesof the invention, the Nanobodies of the invention may generally bedirected against any antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of a metalloproteinase fromthe ADAM family. However, it is generally assumed and preferred that theNanobodies of the invention (and polypeptides comprising the same) aredirected against one of the catalytic sites or binding sites mentionedgenerally herein for the amino acid sequences of the invention.

As already described herein, the amino acid sequence and structure of aNanobody can be considered—without however being limited thereto—to becomprised of four framework regions or “FR's” (or sometimes alsoreferred to as “FW's”), which are referred to in the art and herein as“Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as“Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”,respectively; which framework regions are interrupted by threecomplementary determining regions or “CDR's”, which are referred to inthe art as “Complementarity Determining Region 1” or “CDR1”; as“Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively. Somepreferred framework sequences and CDR's (and combinations thereof) thatare present in the Nanobodies of the invention are as described herein.Other suitable CDR sequences can be obtained by the methods describedherein.

According to a non-limiting but preferred aspect of the invention, (theCDR sequences present in) the Nanobodies of the invention are such that:

-   -   the Nanobodies can bind to a metalloproteinase from the ADAM        family with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²        moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or        less and more preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an        association constant (K_(A)) of 10⁵ to 10¹² liter/moles or more,        and preferably 10⁷ to 10¹² liter/moles or more and more        preferably 10⁸ to 10¹² liter/moles);        and/or such that:    -   the Nanobodies can bind to a metalloproteinase from the ADAM        family with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about 10⁷        M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹, more preferably between        10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷        M⁻¹s⁻¹;        and/or such that they:    -   the Nanobodies can bind to a metalloproteinase from the ADAM        family with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s) and        10⁻⁶ s⁻¹ (providing a near irreversible complex with a t_(1/2)        of multiple days), preferably between 10⁻² s⁻¹ and and 10⁻⁶ s⁻¹,        more preferably between 10⁻³ s⁻¹ and 10⁻⁴ s⁻¹, such as between        10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, (the CDR sequences present in) the Nanobodies of theinvention are such that: a monovalent Nanobody of the invention (or apolypeptide that contains only one Nanobody of the invention) ispreferably such that it will bind to a metalloproteinase from the ADAMfamily with an affinity less than 500 nM, preferably less than 200 nM,more preferably less than 10 nM, such as less than 500 pM.

The affinity of the Nanobody of the invention against ametalloproteinase from the ADAM family can be determined in a mannerknown per se, for example using the general techniques for measuringK_(D). K_(A), k_(off) or k_(on) mentioned herein, as well as some of thespecific assays described herein.

Some preferred IC50 values for binding of the Nanobodies of theinvention (and of polypeptides comprising the same) to ametalloproteinase from the ADAM family will become clear from thefurther description and examples herein.

In a preferred but non-limiting aspect, the invention relates to aNanobody (as defined herein) against a metalloproteinase from the ADAMfamily, which consists of 4 framework regions (FR1 to FR4 respectively)and 3 complementarity determining regions (CDR1 to CDR3 respectively),in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;    or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) against ametalloproteinase from the ADAM family, which consists of 4 frameworkregions (FR1 to FR4 respectively) and 3 complementarity determiningregions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    337;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    549;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    761;    or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to aNanobody (as defined herein) against a ADAMS, which consists of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;    or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) ADAMS, whichconsists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 232 to    250;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 444 to    462;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 656 to    674;    or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to aNanobody (as defined herein) against a ADAM9, which consists of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to    987;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004    to 1007;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024    to 1027;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;    or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) ADAM9, whichconsists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to    987;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 251 to    271 and/or 984 to 987;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004    to 1007;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 463 to    483 and/or 1004 to 1007;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024    to 1027;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 675 to    695 and/or 1024 to 1027;    or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to aNanobody (as defined herein) against a ADAM10, which consists of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;    or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) ADAM10, whichconsists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 272 to    295;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 484 to    507;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 696 to    719;    or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to aNanobody (as defined herein) against a ADAM17, which consists of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to    992;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008    to 1012;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028    to 1032;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;    or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) ADAM17, whichconsists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to    992;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 296 to    312 and/or 988 to 992;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008    to 1012;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 508 to    524 and/or 1008 to 1012;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028    to 1032;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 720 to    736 and/or 1028 to 1032;    or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to aNanobody (as defined herein) against a ADAMTS5, which consists of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 and/or 993;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 and/or 993;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 and/or 993;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 and/or 1013;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 and/or 1013;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 and/or 1013;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 and/or 1033;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 and/or 1033;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 and/or 1033;    or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) ADAMTS5, whichconsists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 and/or 993;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 and/or 993;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 313 to    337 and/or 993;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 and/or 1013;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 and/or 1013;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 525 to    549 and/or 1013;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 and/or 1033;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 and/or 1033;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 737 to    761 and/or 1033;    or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the amino acid sequences of theinvention, when one of the Nanobody of the invention mentioned in one ofthe preceding paragraphs contains one or more CDR1 sequences accordingto b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)    and/or c) is preferably, and compared to the corresponding CDR    according to a), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to b) and/or c) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to a);    and/or-   iii) the CDR according to b) and/or c) may be a CDR that is derived    from a CDR according to a) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Similarly, when one of the Nanobody of the invention mentioned in one ofthe preceding paragraphs contains one or more CDR2 sequences accordingto e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)    and/or f) is preferably, and compared to the corresponding CDR    according to d), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to e) and/or f) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to d);    and/or-   iii) the CDR according to e) and/or f) may be a CDR that is derived    from a CDR according to d) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Also, similarly, when one of the Nanobody of the invention mentioned inone of the preceding paragraphs contains one or more CDR3 sequencesaccording to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)    and/or i) is preferably, and compared to the corresponding CDR    according to g), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to h) and/or i) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to g);    and/or-   iii) the CDR according to h) and/or i) may be a CDR that is derived    from a CDR according to g) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally applyto any Nanobody of the invention that comprises one or more CDR1sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e),f), h) or i), respectively.

Of the Nanobodies of the invention, Nanobodies comprising one or more ofthe CDR's explicitly listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's explicitly listed above are moreparticularly preferred; and Nanobodies comprising three of the CDR'sexplicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table A-1 below, which lists theCDR sequences and framework sequences that are present in a number ofpreferred (but non-limiting) Nanobodies of the invention. As will beclear to the skilled person, a combination of CDR1, CDR2 and CDR3sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3sequences that are mentioned on the same line in Table A-1) will usuallybe preferred (although the invention in its broadest sense is notlimited thereto, and also comprises other suitable combinations of theCDR sequences mentioned in Table A-1). Also, a combination of CDRsequences and framework sequences that occur in the same clone (i.e. CDRsequences and framework sequences that are mentioned on the same line inTable A-1) will usually be preferred (although the invention in itsbroadest sense is not limited thereto, and also comprises other suitablecombinations of the CDR sequences and framework sequences mentioned inTable A-1, as well as combinations of such CDR sequences and othersuitable framework sequences, e.g. as further described herein).

Also, in the Nanobodies of the invention that comprise the combinationsof CDR's mentioned in Table A-1, each CDR can be replaced by a CDRchosen from the group consisting of amino acid sequences that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity (as defined herein) withthe mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table A-1, a    conservative amino acid substitution (as defined herein);    and/or-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table A-1;    and/or-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table A-1.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table A-1 will generally be preferred.

TABLE A-1 CDR's and framework sequences of Nanobodies against fiverepresentative ADAM's (ADAM 8, ADAM 9, ADAM 10. ADAM 17 and ADAM TS5)SEQ FR1 SEQ CDR1 SEQ FR2 SEQ CDR2 SEQ FR3 SEQ CDR3 Clone against ADAM828-B12, 126 EVQLVESGGGL 232 LYTSG 338 WYRQAPG 444 SIASGASGGT 550RFTISRDDAENT 656 QDPMRTR VQNGGSLRLS KQREWVA TVYEDSVKG VYLQMITLKPED DAYCVMSGSSIS TAVYYCRA 28- 127 EVQLVESGGGL 233 NYIMG 339 WFRQAPGK 445AINYEGDRTY 551 RFTISRDNAKNT 657 GPRYGKY D1, H1 VQAGGSLRLSC EREFVASPNSVKG VYLQMNSLKPED DY AASGRTFD TAVYQCAT 28-A6 128 EVQLVESGGGL 234DYTMY 340 WFRQAPGK 446 AIGGVTDYAD 552 RFTIARDNAKST 658 KHRSVATVQAGGSLRLSC ERELVA SVKG VSLQMNSLKPED RTGGNVS AASGRTFS TAVYYCAA 28-B6 129EVQLVESGGGL 235 IRVMG 341 WFRRAPGK 447 TVTWRDNITY 553 RFTITRDNAKNT 659QTEDSAQ VQAGASLKLSC DRESVA YVDSVKG VYLQMNNLKPED YIY AASGRTYD TAVYYCAA28-C1 130 EVQLVESGGGL 236 NYVMG 342 WFRQAPGK 448 AMGDTTLYA 554RFTISRDNDQNT 660 RSRFIPP VQAGGSLRLSC EREFVA DSVEG VYLQMSSLKPED LTFRTGGAASGRTFS TAAYFCAA AYDY 28-C6 131 EVQLVESGGGL 237 IRVMG 343 WFRRAPGK 449TTTWRDNITY 555 RFTITRDNAKNT 661 QTEDSAQ VQAGASLKLSC GRESVA YMDSVKGVYLQMNNLKPED YIY AASGRTYD TAVYYCAA 28-D12 132 EVQLVESGGGL 238 FNAMA 344WHRQAPG 450 AIHISGNTNY 556 RFTISRDNGKNT 662 IEVRTGL VQAGGSLRLSC NQRELVAADSVKG VYLQMNSLRPED SPRGH ATSGSIAS TAVYYCNA 28-E1 133 EVQLVESGGGL 239DYTMA 345 WFRQAPGK 451 AIGSVTDYAD 557 RFTISRDNAKNT 663 KHRTRTRVQAGGSLRLSC ERELVA SVKG VSLQMNSLKPED GYVN TASGRTFS TAVYYCAA 28-E6 134EVQLVESGGGL 240 IETMA 346 WHRQAPG 452 AIRSDDMTNY 558 RFTISRDNFKNT 664IQRRAPY VQAGGSLRLSC KQRELVA PDSVKG VYLQMNSLTPED SRLETY AASGNIFR TAVYYCNL28-F12 135 EVQLVESGGGL 241 FNSMA 347 WYRQSPG 453 RIFRSANIYY 559RFTISRDSALNTV 665 RLSNGLDY VQAGESLRLSC KQRNLVA DDSVKG FLQMNALKSEDTKASRNDFS AVYYCNG 28-G1 136 EVQLVESGGGL 242 IETMA 348 WHRQAPG 454AIRRDEMTNY 560 RFTISRDNFKNT 666 IQRRAPY VQAGGSLRLSC KQRELVA LDFVKGVYLQMNSLKPED SRLETY AASGNIFR TAVYYCNL 28-G12 137 EVQLVESGGGL 243 INTMG349 WYRQTPG 455 SISSGGTTAY 561 RFSISRDGPKNT 667 QRRWSQDY VQAGGSLRLSCQQRDWVA ADSVKG VYLQMNSLKPED AASGSIFS TAVYYCKA 28-G6 138 EVQLVESGGGL 244FNAVG 350 WWRQAPG 456 SISTSGSTITP 562 RFTISKDNTKNT 668 TERMHHSYAQAGGSLRLSC TQREWVA YVDSVKG VYLQMNSLKPED AASGSITS TAVYYCCA 29-A12 139EVQLVESGGGL 245 FWMG 351 WFRQTPG 457 AISGSFNVTS 563 RFTISRDNAKNT 669QRWRGGSY VQAGGSLRLSC MKRFFVA YADSVKG VTLQMNSLKPFD FY AASGRTFS TAVYYCAA29-A6 140 FVQLVFSGGGL 246 YDAMA 352 WFRQAPGK 458 VITWSGGTTV 564RFTISRDIAKNTM 670 SAGARYGV VQAGGSLGLS FRFFVA YADSVQG NLQMNSLKPDDTGWWRNGQNY CVFSGRPYS AVYYCAG QN 29-C6 141 FVQLVFSGGFS 247 NFIMG 353WFRRAPGK 459 AIGDPSTHYA 565 RFTISRDNAKNM 671 RSRYSTGT VQVGGSVRLS FRFLLADSATG VYLQMNSLKTFD LYDQTKYI CAASGLTFS TAVYYCAA Y 29-F12 142 FVQLVFSGGGL248 RHVIG 354 WFRQAPGK 460 AINSDGDTTT 566 RFTISRFNANNT 672 GPQYRSYFVQAGGSLTLSC FRKFVT DDRSLKG VHLQMTNLKVFD ARSYLY WSGVDFS TAIYYCAT 29-F6143 FVQLVFSGGG 249 DYIIG 355 WFRQGPG 461 RISGSGLTNT 567 RFTISRDNAKNT 673AYSGHFSGR RVQAGGSLRLS KFRFSVA TTFTVKG VYLQMNSLKPFD VSDFLY CFASGRTFSTAVYYCAA 29-H1 144 FVQLVFSGGGL 250 IHAMG 356 WYRQAPG 462 SITSSATAYA 568RFIISRDNAENTI 674 QVLVPGGR VQPGGSLRLAC NFRFWVA DSVKG YLQMHSLKPFDT NDYAASKSIDN AVYYCKG Clone against ADAM9 41- 145 EVQLVESGGGL 251 TYAMG 357WFRQAPGK 463 AISWNEVNTY 569 RFTISRNNAENT 675 DRHYTAQQ B5, C1, D4VQAGGSLRLAC EREFVA YTDSVKG VYLQMNSLKRED MRVMTGAS 3, E4, E5 AASGRTFSTAVYYCAS YMDY 30-B12, 146 EVQLVESGGGL 252 IYDMG 358 WFRQAPGK 464VIRWSDGFTY 570 RFTISRDNAKNT 676 NTAPLRII VQAGGSLRLSC EREFVT YEDSVKGVYLQMNNLKPED NFRNAYNY AASGPTFS TAVYVCAA DY 30-A6 147 EVQLVESGGGL 253DYAMG 359 WFRQAPEK 465 TINYSGGVTY 571 RFTISRDNAKNT 677 DRHYGAYAVQAGGSLRLSC EREFVA YADSVKG VYLQMNSLKPED LLGLGTHA AASGRASG TAVYYCAA YIDY30-B1 148 EVQLVESGGGL 254 RLAMG 360 WFRQAPGK 466 AINWLSESTY 572RFTISRDNAKNT 678 DRFATAQN VPAGGSLRLSC ERDFVG YEDSVKG VYLQMNSLKPEDMNTMGSLD AGSGRSFS TAVYYCAS Y 30-C1 149 EVQLVESGGGL 255 SYAVG 361WFRQAPGK 467 VISWTGGSTY 573 RFTISRDNAKNT 679 DRFTTGSG VQAGDSLRLSC EREFVAFADSVKG VYLQMNNLKSED RTSYPRPS AVSGRTIS TAVYYCTA EFDH 30-D1 150EVQLVESGGGL 256 SDWMY 362 WVRQAPG 468 SIGIAGTPTFY 574 RFTISRDNANNM 680EGIYCSNW VQPGGSLRLSC KGPEWVS ADSVKG LYLQMTSLKPGD RCLFGPKT AASGFAFSTALYYCAR DLPAS 30-D12 151 EVQLVESGGGL 257 DYWG 363 WFRQTPGK 469RKVWSNGNT 575 RFTISGDNAKRT 681 RSPMSPTW VQAGGSLRLSC EREFIG YYIDSVKGVYLQMNSLKPED DN ATSGRTFS TALYYCAA 30-E12 152 EVQLVESGGGL 258 RLAMG 364WFRQAPGK 470 AISWLGESTY 576 RFTISRDNAKNT 682 DRFATAQA VPAGGSLRLSC EREFVAYDDSVKG VYLQMNSLKPED MGAMGSLD AGSGRTFS TAVYYCAS Y 30-G1 153 EVQLVESGGGL259 NYFMG 365 WFRQAPGK 471 GVAWSSDFT 577 RFTISRDNAKNT 683 RRRGGGYNVQAGISLKLSC EREFVA AYTDSVKG VYLQMNDVKPE QLNLYDY TASGGTLT DTAVWYCAA30-G12 154 EVQLVESGGRL 260 TDVMG 366 WFRQVPEK 472 EIGWRDTTLY 578RFTISRDNAKNM 684 RRAAAAYDQ VQAGGSLRLSC EREFVA ADSVKG VYLQMNSLKPEDAASGRTFS TAVYYCSS 31-B1 155 EVQLVESGGGL 261 TLAMG 367 WFRQAPGK 473AISWSEVNTY 579 RFTISRNNAENT 685 DRHYSAQQ VQAGGSLRLAC EREFVA YTDSVKGVYLQMNSLKPED MRVMTGAS AASGRTFS TAVYYCAA YMDY 31-D12 156 EVQLVESGGGL 262NYHVG 368 WFRQAPGK 474 AISASGTSTA 580 RFTISRDNAKNT 686 SEYVFRYYVQAGDSLRLSC EREFVA YAGSVKG AFLQMNTLKPED DDSRYYAY APSGGIIS TAVYYCAA31-E12 157 EVQLVESGGGL 263 AYNMG 369 WFRLRPGK 475 AINWSGGTQ 581RFTAIADVAKKTV 687 TQWGSSGW VQAGGSLRLSC EREFVA DYVDSVKG FLQMTSLKPEDTKQARWYDF SSSGRTYS AVYYCAA 31-F6 158 EVQLVESGGGL 264 TLAMG 370 WFRQAPGK476 AISWSEVNTY 582 RFTISRNNAENT 688 DRHYTAQQ VQAGGSLRLAC EREFVA YTDSVKGVYLQMNSLKRED MRVMTGAS AASGRTFS TAVYYCAS YMDY 31-G12 159 EVQLVESGGGL 265NYVMG 371 WFRQAPGK 477 AITWSDSRTD 583 RFTISRDNVKNL 689 SSIGPYRLVQAGESLRLSC ERELVA YADSVKG VYLQMNSLRPED LDSSRYAY AASGRTFG TAVYSCAA 41-A1160 KVQLVESGGGL 266 TLAMG 372 WFRQAPGK 478 AISWSEVNTY 584 RFTISRNNAENT690 DRHYSAQQ VQAGGSLKLAC FRFFVA YTDSVKG VYLQMNSLKPED MRVMTGAS AASGRTFSTAVYYCAA YMDY 41-A3 161 FVQLVFSGGGL 267 TYAMG 373 WFRQAPGK 479AISRNFVNTY 585 RFTISRNNAENT 691 DRHYTAQQ VQAGGSLRLAC FRFFVA YTDSVKGVYLQMNSLKRFD MRVMTGAS AASGRTFS TAVYYCAS YMDY 41-B1 162 FVQLVFSGGGL 268GYHMG 374 WFRQAPGK 480 YISSGGAYSN 586 RFTISRDNAKNT 692 TDYNKAYAVQAGGSLRLSC FRFFVA YADSVKG AYLQMNSLKPFD RFGRRYDY TGSDRTFI TAVYHCAA 41-C5163 FVQLVFSGGGL 269 AVFMG 375 WYRQLPGK 481 SINRGGSTNY 587 RFTISRVNANST693 VINTRSF VQAGGSLRLSC QRFLVA APSAKG MYLQMNSLQPF WSGTITS DTAVYYCYG41-D1 164 FVQLVFSGGGL 270 TLAMG 376 WFRRAPGK 482 AISWSFDNTY 588RFTISRNNAEST 694 DRHLLAQQMR VQAGGSLRLAC FRFFVA YSDSVKG VYLQMNSLKPFDVMTGASYMDY AASGRTFS TAVYYCAS 41-F6 165 FVQLVFSGGGL 271 TLAMG 377WFRQAPGK 483 AISWSFVNTY 589 RFTISRNNAENT 695 DRHATAQHMR VQAGGSLRLACFRFFVA YTDSVKG VYLQMNSLKPFD VMTGASYMDY AASGRTFS TAVYYCVS 30-B6 974EVHLVESGGG 984 EYALV 994 WFRQAPGK 1004 VAGFAANGIN 1014 RFTISRDNAKNT 1024SWTPAYNDIPR WQAGGSLRLS EKEREF TDYTDSVKG VYLQMNSLKPED HMSSMDQ CVASGLPFSTAVYYCAA 30-C6 975 EVQLVESGGGL 985 SNTMA 995 WYRQAPRK 1005 SIMTDGTITY1015 RFAISRDTAKNT 1025 RQYGEYWQAA VQAGGSLRLSC QREFVA ADSVKG VALQMNSLKPEDGS AASGSIFS TAVYYCNA x30-D6 976 EVQLVESGGGL 986 RLAMG 996 RWFRQAPGK 1006AISWLAETTY 1016 RFTISRDNAKNT 1026 DRFATAQHMG VQPGGSLRLSC EREFVA YEDSVKGVYLQMNSLKPED AMGSLDY AGSGRTFS TAVYYCAS 30-F12 977 EVQLVESGGGL 987 SYAIA997 WFRQAPGK 1007 CISSSDGTTY 1017 RFTISRDNAKNM 1027 DPELVTVCRPGVQPGGSLRLSC EREGVS YADSVKG VYLQMQSLKPED WGPAYDY AASGFTLD TAVYYCAA Clonesagainst ADAM10 39-A8, 166 EVQLVESGGGL 272 SYCVG 378 WWRQAPG 484AITRGSNSTD 590 RFTISRDNAENT 696 DINCRNLYTGR D7, E7, VQTGGSLRLSC KERDWAYVDSVKG VYLQMNSLKPED PEY F9, G8, AASGRTFT TAVYYCAA H8, A9 32- 167EVQLVESGGGL 273 DYAIG 379 WFRQAPGK 485 CISISDSSTYY 591 RFTISSDNAKNT 697DRLAYGLDPNF E12, G12 VQAGGSLRLSC EREGVS ADSVKG VYLQMNSLKPED YDY AASGFTFGTAVYYCAA 32- 168 EVQLVESGGGL 274 TYFMG 380 WFRQAPGK 486 FISGNGGSTD 592RFAISRDNVKNT 698 AGRQIKSTWD E1, E6, VQAGGSLRLSC EREFVA YADSVKGLYLQMSSLKPDD Y AASERIFS TAVYYCAV 32-D1 169 EVQLVESGGGL 275 DYAIG 381WFRQAPGK 487 CISVSDGSTY 593 RFTISSDNAKNT 699 DRLAYGLDPNF VQAGGSLRLSCEREGVS YADSVKG VYLQMNSLKPED YDH AASGFTFD TAVYYCAA >39- 170 EVQLVESGGGL276 RYAMG 382 WFRQAPGK 488 AISRSGGNTY 594 GRFTMSRDNAK 700 RSAAVYTTTVYD8, E8 VRARGSLRLSC EREFVA FTESVK NTVYLQMNSLKP LVDFEYNY AASGGTFSEDTAVYYCAA 32-06 171 KVQLVESGGGL 277 DYAIG 383 WFRQAPGK 489 CISISDSSTYY595 RFTISSDNAKNT 701 DRLAYGLDPNF VQAGGSLRLSC EREGVS ADSVKG VYLQMNSLKPEDYDY AASGFTFG TAVYYCAA 32-F1 172 EVQLVESGGGL 278 SYRLG 384 WFRQAPGN 490SITWSSANTY 596 RFTISRERAKNT 702 EDVGKPFDS VQAGGSLTLSC EREFVA YADSVKGMYLQMDSLRPE AASGRTFS DTAVYYCAK 32-G6 173 EVQLVESGGGL 279 DYAIG 385WFRQAPGK 491 CISSSDGRTY 597 RFTISSDNAKNT 703 DRLAYGLDPNF VQPGGSLRLSCEREGVS YDDSVKG VYLQMNSLKPED YDY AASGFTFD TAVYYCAA 33-A1 174 EVQLVESGGGL280 RYSMG 386 WFRQAPGK 492 YITRSGRTTY 598 RFTISRDNAKNT 704 MRSGNVRYPEVQAGGSLRLSC EREFW YQDSVKG VYLQMNSLKPED RYDY AASGDTFS TAVYYCAA 33-C6 175EVQLVESGGGL 281 SYYMG 387 WFRQAPGK 493 HISLSGGNTE 599 RFTITRDNAGNT 705SPSLRSAWQY VQAGDSLRLSC EREFVA YADSVKG VYLQMNSLKPED AASGRTFS TGVYCCAA33-D1 176 EVQLVESGGGL 282 SDAMG 388 WFRQAPGK 494 GINYNSVYRY 600RFTISRDNAKNT 706 DSDYYSLIGGR VQAGDSLRLSC ERAFVA YTDSVEG VYLQMNSLKPEDPVNY AATGRTFS TAVYYCAA 33-G6 177 EVQLVESGGG 283 SYCVG 389 WWRQAPG 495AITRGGDTTD 601 RFTISRDKAENT 707 DINCRNLYTGR SVQTGGSLRLS KERAWA YVDSVKGVYLQMNSLKPED PEY CAASGRTFT TAVYYCAA 39-B1 178 EVQLVESGGGL 284 INIMA 390WYRQAPG 496 AIIGDSTNYA 602 RFTISRDNAKNT 708 SGVD VQAGGSLRLSC KTRDMVADSVKG VYLHMNRLKPED ARSGRISN TGVYYCKI 39-B2 179 EVQLVESGGGL 285 DYAIG 391WFRQAPGK 497 CISSSDGRTY 603 RFTISSDNAKNT 709 DRLAYGLDPNF VQAGGSLRLSCEREGVS YADSVKG VYLQMNSLKPED YDY AASGFTFD TAVYYCAA 39-C1 180 EVQLVESGGGL286 SYVMG 392 WFRQAPGK 498 AVSRSGRNIN 604 RFTVSRDNTKNT 710 DGTISSSWADLVQAGGSLRLSC EREEVA YADLMKG VYLQLNNLTPED RRGETYGD SASGRTFG TAVYYCAA 39-C2181 KVQLVESGGGL 287 RYYMG 393 WFRQAPGK 499 FISGTGGSID 605 RFTISRDSAKNT711 ASGNSGRSTW VQAGGSLRLSC EREEVA YADSVKG VYLQMNSLKPED DY AASGRTFSTAVYYCAA 39-E1 182 EVQLVKSGGGL 288 NNAVG 394 WYRQAPG 500 AMLSGGSTN 606RFTISRDNAKNT 712 QVNGIWAR VQAGGSLRLSC KQREMVA YADSVKG VYLQMNSLKPEDAASGNIFI TAVYYCNV 39-E2 183 EVQLVESGGGL 289 INIMS 395 WYRQAPG 501AIIGDATNYA 607 RFTISRDNAKNA 713 PGVD VQAGGSLRLSC KTRDMVA DSVKGVSLHMNRLKPED GRSGRISN TGVYYCKI 39-F1 184 EVQLVESGGGL 290 TYFMG 396WFRQAPGK 502 FISGNGGSTD 608 RFAISRDNVKNT 714 AGRQIKSTWG VQAGGSLRLSCEREFVA YADSVKG LYLQMSSLKPDD Y AASERIFS TAVYYCAV 39-G1 185 EVQLVESGGGL291 INIMS 397 WYRQAPG 503 AIIGDSTNYA 609 RFTISRDNAKNT 715 PGVDVQAGGSLRLSC KTRDMVA DSVKG VHLQMNRLKPE ARSGRISN DTGVYYCNI 39-G2 186EVQLVESGGGL 292 DYAIG 398 WFRQAPGK 504 CISMSDGSTY 610 RFTISSDNAKNT 716DRLAFGLDSNF VQAGGSLRLSC EREGVS YADSVKG VYLQMNSLKPED YDY AASGFTFDTAVYYCAA 39-C8 187 EMQLVESGGG 293 SYCVG 399 WWRQAPG 505 AITRGSNSTD 611RFTISRDNAENT 717 DINCRNLYTGR LVQTGGSLRLS KERDWA YVDSVKG VYLQMNSLKPED PEYCAASGRTFT TAVYYCAA 39-D9 188 EVQLVESGGGL 294 SYCVG 400 WWRQAPG 506AITRGSNSTD 612 RFTISRDNAENT 718 DINCRNLYTGR VQPGGSLRLAC KERDVVA YVDSVKGVYLQMNSLKPED PEY AASGRTFT TAVYYCAA 39-01 189 KVQLVESGGGL 295 NNAVG 401WYRQAPG 507 AMLSGGSTN 613 RFTISRDNAKNT 719 QVNGTNAR VQAGGSLRLSC KQREMVAYADSVKG VYLQMNSLKPED AASGNIFI TAVYYCNV Clones against ADAM17 35- 190EVQLVFSGGGL 296 TYAMG 402 WFRQAPGK 508 GITRSAVSTS 614 RFTISRDNAKDM 720TRRLHFGNGA B12, E1, VQTGGSLRLSC ERFFVA YTDSVKG VYLQMNSLKPED DY G1, F6AASGGTFS TALYYCAA 35-C6, 191 KVQLVFSGGGL 297 TYAMG 403 WFRQAPGK 509GITRSAVSTS 615 RFTISRDNAKDM 721 TRRLHFGNGA VQTGGSLRLSC FRFFVA YTDSVKGVYLQMNSLKPFD DY AASGGTFS TALYYCAA 40- 192 EVQLVKSGGGL 298 TYAMG 404WFRQAPGK 510 GITRSGLSTS 616 RFTISRDNAKDT 722 TRRLHFGNGA A2, H2VQTGGSLRLSC EREFVA YADSVKG VYLQMNSLKPED DY IVSGGTFS TALYYCAA 40- 193EVQLVESGGGL 299 TYAMG 405 WFRQAPGK 511 GITRSGLSTS 617 RFTISRDNAKDT 723TRRLHFGNGA B1, D1 VQTGGSLRLSC EREFVA YADSVKG VYLQMNSLKPED DY IVSGGTFSTALYYCAA 35- 194 EVQLVKSGGGL 300 TYAMG 406 WFRQAPGK 512 GITRSGVSTS 618RFTISRDNAKDM 724 TRRLHFGNGA B1, G12 VQTGGSLRLSC FRFFVA YADSVKGVYLQMNSLKPFD DY AASGGTFS TALYYCAA 35-C12 195 KVQLVESGGGL 301 TYAMG 407WFRQAPGK 513 GITRSAVSTS 619 RFTISRDNAKDM 725 TRRLHFGNGA VQTGGSLRLSCEREFVA YTDSVKG VYLQMNSLKPED DY AASGGTFS TALYYCAT 35-F12 196 EVQLVESGGGL302 TYAMG 408 WFRQAPGK 514 GITRSGVSTS 620 RFTISRDNAKDM 726 TRRLHFGNGAVQTGGSLRLSC EREEVA YADSVKG VYLQMNSLKPED DY AASGGTFS TALYSCAA 35-G6 197EVQLVESGGGL 303 YAIA 409 WFRQTPGK 515 VINRSGSYVY 621 RFTISRHNAKNT 727RDGTLYSTTYY VQAGDSLRLSC EREFVA YTDSVKG AYLQMNSLKPED YISSYTY TASGAFGSTAVYYCAA 40-A1 198 EVQLVESGGGL 304 TYAMG 410 WFRQAPGK 516 GITRSAVSTS 622RFTISRDNAKDM 728 TRRLHFGNGA VQAGGSLRLSC EREEVA YTDSVKG VYLQMNSLKPED DYAASGGTFS TALYYCAA 40-B2 199 EVQLVESGGGL 305 TYAMG 411 WFRQAPGK 517GITRSALSTS 623 RFTISRDNAKDM 729 TRRLHFGNGA VQTGGSLRLSC FREEVA YADSVKGVYLQMNSLKPED DY IASGGTFS TALYYCAA 40-C1 200 FVQLVFSGGGL 306 YAIA 412WFRQTPGK 518 VINRSGSYVY 624 RFTISRHNAKNT 730 RDGTLYSTTYY VQAGDSLRLSCFREFVA YTDAVKG AYLQMNSLKPED YISSYTY TASGTFGS TAVYYCAA 40-D2 201FVQLVFSGGGL 307 KLAVA 413 WFRQAPGK 519 GITRSAVSTS 625 RFTISRDNAKDM 731TRRLHFGNGA VQPGGSLRLSC KLAVA YTDSVKG VYLQMNSLKPED DY RASGGTFR TALYYCAA40-F1 202 FVQLVFSRGGL 308 TYAMA 414 WFRQAPGK 520 GITRSAVSTS 626RFTISRDNAKDM 732 TRRLHFGNGA VQTGGSLRLSC EREFVA YADSVKG VYLQMNSLKPED DYAASGGTFS TALYYCAA 40-F2 203 FVQLVFSGGGL 309 TYAMG 415 WFRQAPGK 521GITRSSVSTS 627 RFTISRDNAKDM 733 TRRLHFGNGA VQTGGSLRLSC FREFVA YADSVKGVYLQMNSLKPFD DY AASGGTFS TALYYCAA 40-G1 204 EVQLVESGGGL 310 YAIA 416WFRQTPGK 522 VINRSGSYMY 628 RFTISRHNAKNT 734 RDGTLYSTSYY VQAGDSLRLSCEREFVA YIDSVKG AYLQMNSLKPED YISSYTY TASGTFGS TAVYYCAA 40-G2 205EVQQVESGGG 311 TYAMG 417 WFRQAPGK 523 GITRSGVSTS 629 RFTISRDNAKDT 735TRRLHFGNGA LVQTGGSLRLS EREFVA YADSVKG VYLQMNSLKPED DY CIVSGGTFS TALYYCAA40-H1 206 EVQLMESGGG 312 TYAMG 418 WFRQAPGK 524 GITRSAVSTS 630RFTISRDNAKDM 736 TRRLHFGNGA LVQTGGSLRLS EREFVA YTDSVKG VYLQMNSLKPED DYCAASGGTFS TALYYCAA 34-A12 978 EVQLVESGGGL 988 YYAIG 998 WFRQAPGK 1018CISSSDGIRY 1028 RFTISRDNAKNM 1028 DPIRICSSQPR VQPGGSLRLSC EREGVS YIDSVKGVYLQMNSLKPED RYDY AASGFPLD TAVYYCAA 34-H1 979 EVQLVESGGGL 989 SYVMG 999WFRQAPGK 1019 TISRSGESTY 1029 RFTISRDNAENT 1029 DYNPYQSGSY VQDGGSLSCAEREFVA YTGSVKG VYLQMNSLKPED YSSRSSTYDY ASGRTFS TAVYYCAA 35-A12 980EVQLVESGGGL 990 DYAIG 1000 WFRQAPGK 1020 CIGDKGDRIF 1030 RFAISSDHAKHT1030 VAPFAFCTESL VQAGGSLRLSC EREGVS YADSVKG VDLQMTNLKPED PDTWYDYVASGLSFD TATYYCAA 35-D1 981 EVQLVESGGGL 991 TYAMA 1001 WFRRAPGK 1021AIRWSGDRT 1031 RFTISRDNAKNT 1031 RRFSGFDYSG VQAGGSLRLSC EREFW YYADSVKGVYLQMNSLKPED NYYAWDAYDY AASGRTFS TAVYYCAL 35-H1 982 EVQLVESGGG 992 SYAMG1002 WFRQAPGK 1022 GINGSGNGIR 1032 RFTITRDNAKNT 1032 TFAAGASEYHYWVQAGDSLRL EREFVA IALSVRG GYLQMNSLKRE SCAASGRTFS DTAVYYCGP Clone againstADAM TS5 36-A6, 207 EVQLVESGGGL 313 SYNMG 419 WFRQAPGK 525 ADMWSGTTT 631RFTISRDNAKNM 737 ELHSSDYTSPG 40-H8 VQAGDSLRLSC EQEFVA YYTDSVKGVYLQMNSLKPED AYAY AASGRTLS TAVYYCAA 36-A1 208 EVQLVESGGGL 314 IRAMG 420WLRQAPGN 526 AISRDGDRTY 632 RFTISRDNAKST 738 TRPFKVLTATI VQPGGSLTLSCEREFVA YTDWKG VYLQINSLKTEDT ENDFTY AASGGTFS AVYYCAA 36-06 209EVQLVESGGGL 315 SRYHMG 421 WFRQGPG 527 AVSSLGPFTR 633 RFTISRDNAKNA 739DSSGYSGSYS VQAGGSLRLSC KEREFVA YADSVKG VYLQMNSLKPED SEYRYDY AFSDGRTVTAVYYCAA 36-D6 210 EVQLVESGGGL 316 NYAMA 422 WFRQAPGK 528 GINWSGNGV 634RFAISRENPKNM 740 DRTLTAWDRD VQAGGSLRLSC EREFVA YYPDSLKE VYLQMNSLNPEDNAEY VASGLTFR TAVYYCTA 36-E1 211 EVQLVESGGGL 317 SYNMG 423 WFRQAPGK 529AIMWSGTTTY 635 RFTISTDNAKNM 741 ELHSSDYTSPG VQAGDSLRLSC ELEFVA YTDSVKGVYLQMNSLKPED AYAY AASGRTLS TAVYYCAA 36-F1 212 EVQLVESGGGL 318 IRAMG 424WFRQAPGN 530 AISRDGDRTY 636 RFTISRDNAKST 742 TRPFKVLSAIIE VQPGGSLTLSCEREFVA YTDWKG VYLQINSLKTEDT NDFTY AASGGTFS AVYYCAA 37-B1 213 EVQLVESGGGL319 PYKVA 425 WFRQAPGK 531 VIHWYGITAY 637 RFSISRDNAKGT 743 DSTSALGHATTVQPGQSLRLSC ERDFVA ADTVKG VYLQMDSLKPED DFDS AASGRTLN TAVYYCAL 37-B12 214EVQLVESGGGL 320 NYHMA 426 WFRQAPGK 532 DFRPSGGSP 638 RFTIFRDNAKNT 744DSHGGIAFMEP VQAGGSLRLSC EQEFVG YYANYADSV VYLQMNSLKLED DEYDY AASGRSLR KGTAVYYCAA 37-B6 215 EVQLVESGGGL 321 IYPIG 427 WFRQAPGK 533 AIDWSGSRS 639RFTISGDNAKNT 745 RPVGDFADPR VQAGGSLRLSC EREFVA YYLDSMKG VYLQMNSLKPED YRFTAAGRTHS TGVYYCAA 37-C12 216 EVQLVESRGGL 322 GYGVG 428 WFRQGPG 534AISWSPGRT 640 RFTISRDNAKNT 746 DSVSASYYDA VQTGGSLRLSC KDREFVA DYGDAVKGVYLQMNSLKPED RNDMAYDY TTSGGTLR TAVYYCAA 37-06 217 EVQLVESGGG 323 NYGVG429 WFRQGPG 535 AISWSPGRT 641 RFTISRDNPKNT 747 DSVSASYYDA WQTGGSLRLSKDREFVA DYGDAVKG VYLQMNSLKPED RNDMAYDY CTTSGGTFR TAVYYCAA 37-D6 218EVQLVESGGGL 324 PYKVA 430 WFRQAPGK 536 VIHWYGITAY 642 RFSISRDNAQGT 748DSTSALGHTTS VRAGGSLRLSC ERDFVA ADTVKG VKLQMDSLKPED DFDS AASGRTLNTAVYYCAL 37-E6 219 EVQLVESGGGL 325 TYHMA 431 WFRQAPGK 537 DLRPSGGRA 643RFTIFRDNAKNT 749 DSHGGISFMEP VQAGGSLRLSC EQEFVG GYADYADSV VYLQMNSLKLEDDEYDY AASGRSLG KG TAVYYCAA 37-F1 220 KVQLVESGGGL 326 PYKVA 432 WFRQAPGK538 VIHWYGITAY 644 RFSISRDNAKGT 750 DSTSALGHATT VRAGGSLRLSC ERDFVAADTVKG VYLQMDSLKPED DFDS AASGRTLN TAVYYCAL 37-F12 221 EVQLVESGGGL 327NYGVG 433 WFRQGPG 539 AISWSPGRT 645 RFTISRDNAKNT 751 DSVSASYYDAVQTGGSLRLSC KDREFVA DYGDAVKG VYLQMNSLKPED RNDMAYDY TTSGGTFR TAVYYCAA37-G1 222 EVQLVESGGGL 328 MGRVG 434 WFRQDPGK 540 AISRSGDTTY 646RFTTSRDNAKNT 752 ATFRAVRQDP VQAGGSLRLTC EREFVA YDDSVKD IDLRMNSLKPEDSYSASYDY AASGRTFS TAVYYCAA 37-G6 223 EVQLVESGGGL 329 PYKVA 435 WFRQAPGK541 VIHWYGITAY 647 RFSISRDNAKGT 753 DSTSALGHATT VRAGGSLRLSC ERDFVAADTVKG VYLHMDSLKPED DFDS AASGRTLN TAVYYCAL 40-A7 224 EVQLVESGGGL 330MYTMG 436 WFRQAPGK 542 AITPINWGGR 648 RFTIFRDNTKNTI 754 ESHGSTSPRNVQAGASLRLSC ERDFVA GTHIADSVKG NLQMNNLNPED PLQYDY GASGRTLS TAVYYCAA 40-B8225 EVQLVESGGGL 331 FTNYAIA 437 WFRQAPGK 543 WNWSTYYA 649 RFTISRDNAKNT755 GDYRSDYRSP VQAGGSLRLSC ERKFVA DSVKG VNLQMNSLKPED VAYNY AASDSARTTAVYYCAV 40-D7 226 EVQLMESGGG 332 SYTMA 438 WFRQAPGK 544 YVFGGGEITD 650RFTISKDNAKNT 756 ECVGDVYRSR LVQAGGSLRLS EREEVA YADFVKG GYLQMNSLKPE DYTYCAASGRTIS DTAVYYCAM 40-F7 227 EVQLVESGGGL 333 NYAMA 439 WFRQAPGK 545GINWSGNGV 651 RFAISRENAKNM 757 DRTLTAWDRD VQAGGSLRLSC EREEVA YYPDSLKEVYLQMNSLNPED SAEY VASGLTFR TAVYYCTA 40-F8 228 EVQLVESGGGL 334 MYGSG 440WFRRAPGK 546 FINHSGGRTN 652 RFTISRDNAKDT 758 PISGYINPAVY VQAGGSLRLSCEREFVG YADSVKG VYLQMNSLKPED DRPGSYDY AASGLTFR TAVYYCAV 40-G7 229EVQLVESGGGL 335 NYAMA 441 WFRQAPGK 547 GINWSGNGV 653 RFAISRENSKNM 759DRTLTAWDRE VQAGGSLRLSC EREFVA YYPDSLKE VYLQMNSLNPED SAEY VASGLTFRTAVYYCTA 40-G8 230 EVQLVESGGGL 336 MYTMG 442 WFRQAPGK 548 AITPINWGGR 654RFTIFRDNTKNTI 760 ESHGSTSPRN VQAGASLRLSC ERDFVA GTHIADSVKG ILQMNNLNPEDTAPLQYDY GASGRTLN VYYCAA 40-H7 231 EVQLVESGGGL 337 SYTMA 443 WFRQAPGK 549YVFGGGEITD 655 RFTISKDNAKNT 761 ECVGDVYRSR VQAGGSLGLS EREFVA YADFVKGGYLQMNSLKPE DYTY CAASGRTIS DTAVYYCAM x40-E8 983 EVQLVESGGTL 993 AYGTG1003 WFRQAPGK 1013 TISWSGSTTS 1023 RFTVSRDNAKNT 1033 NRYGLVYKEEVXAGGSLRLSC EREFVA YADSVKG VYLQMNSLKPED RHYDF AASGLAFN TAIYYCAM SEQ FR4Clone against ADAM8 28-B12, 762 WGQGTQVTVSS 28- 763 WGQGTQVTVSS D1, H128-A6 764 WGQGTQVTVSS 28-B6 765 WGQGTQVTVSS 28-C1 766 WGQGTQVTVSS 28-C6767 WGQGTQVTVSS 28-D12 768 WGQGTQVTVSS 28-E1 769 WGQGTQVTVSS 28-E6 770WGQGTQVTVSS 28-F12 771 WGQGTQVTVSS 28-G1 772 WGQGTQVTVSS 28-G12 773WGQGTQVTVSS 28-G6 774 CGQGTQVTV 29-A12 775 WGQGTQVTVSS 29-A6 776WGQGTQVTVSS 29-C6 777 WGQGTQVTVSS 29-F12 778 WGQGTQVTVSS 29-F6 779WGQGTQVTVSS 29-H1 780 WGQGTQVTVSS Clone against ADAM9 41- 781WGKGTLVTVSS B5, C1, D4 3, E4, E5 30-B12, 782 WGQGTQVTVSS 30-A6 783RGQGTQVTVSS 30-B1 784 WGQGTQVTVSS 30-C1 785 WGQGIQVTVSS 30-D1 786WGQGTQVTVSS 30-D12 787 WGQGTQVTVSS 30-E12 788 WGQGTQVTVSS 30-G1 789WGQGTQVTVSS 30-G12 790 WGQGTQVTVSS 31-B1 791 WKGKTLVTVSS 31-D12 792WGQGTQVTVSS 31-E12 793 WGQGTQVTVSS 31-F6 794 WGKGTLVTVSS 31-G12 795WGRGTQVTVSS 41-A1 796 WGKGTLVTVSS 41-A3 797 WGKGTLVTVSS 41-B1 798WGQGTQVTVSS 41-C5 799 WGQGTQVTVSS 41-D1 800 WGKGTLVTVSS 41-F6 801WGKGTLVTVSS 30-B6 1034 WGKGTLVTVSS 30-C6 1035 WGQGTQVTVSS x30-D6 1036WGQGTQVTVSS 30-F12 1037 WGQGTQVTVSS Clones 803 WGQGTQVTVSS againstADAM10 39-A8, D7, E7, 804 WGQGTQVTVSS F9, G8, H8, A9 32- 805 WGQGTQVTVSSE12, G12 32- 806 WGQGTQVTVSS E1, E6, 32-D1 807 WGQGTQVTVSS >39- 808WGQGTQVTVSS D8, E8 32-C6 809 WGQGTQVTVSS 32-F1 810 WGQGTQVTVSS 32-G6 811WGQGTQVTVSS 33-A1 812 WGQGTQVTVSS 33-C6 813 WGRGTQVTVSS 33-D1 814WGQGTQVTVSS 33-G6 815 WGQGTQVTVSS 39-B1 816 WGQGTQVTVSS 39-B2 817WGQGTQVTVSS 39-C1 818 WGQGTQVTVSS 39-C2 819 WGQGTQVTVSS 39-E1 820WGQGTQVTVSS 39-E2 821 WGQGTQVTVSS 39-F1 822 WGQGTQVTVSS 39-G1 823WGQGTQVTVSS 39-G2 824 WGQGTQVTVSS 39-C8 825 WGQGTQVTVSS 39-D9 824WGQGTQVTVSS 39-G1 825 WGQGTQVTVSS Clones against ADAM17 35- 826WGKGTPVTVSS B12, E1, G1, F6 35-C6, 827 WGKGTPVTVSS 40- 828 WGKGTPVTVSSA2, H2 40- 829 WGKGTPVTVSS B1, D1 35- 830 WGKGTPVTVSS B1, G12 35-C12 831WGKGTPVTVSS 35-F12 832 WGKGTPVTVSS 35-G6 833 WGQGTQVTVSS 40-A1 834WGKGTPVTVSS 40-B2 835 WGKGTPVTVSS 40-C1 836 WGQGTQVTVSS 40-D2 837WGKGTPVTVSS 40-F1 838 WGKGTPVTVSS 40-F2 839 WGKGTPVTVSS 40-G1 840WGQGTQVTVSS 40-G2 841 WGKGTPVTVSS 40-H1 842 WGKGTPVTVSS 34-A12 1038WGQGTQVTVSS 34-H1 1039 WGQGTQVTVSS 35-A12 1040 WGQGIQVTVSS 35-D1 1041WGQGTQVTVSS 35-H1 1042 WGQGTQVTVSS Clone against ADAM TS5 36-A6, 843WGQGTQVTVSS 40-H8 36-A1 844 WGQGTQVTVSS 36-C6 845 WGQGTQVTVSS 36-D6 846WGQGTQVTVSS 36-E1 847 WGQGTQVTVSS 36-F1 848 WGQGTQVTVSS 37-B1 849WGQGTQVTVSS 37-B12 850 WGQGTQVTVSS 37-B6 851 WGQGTQVTVSS 37-C12 852WGRGTQVTVSS 37-C6 853 WGRGTQVTVSS 37-D6 854 WGQGTQVTVSS 37-E6 855WGQGTQVTVSS 37-F1 856 WGQGTQVTVSS 37-F12 857 WGRGTQVTVSS 37-G1 858WGQGTQVTVSS 37-G6 859 WGQGTQVTVSS 40-A7 860 WGQGTQVTVSS 40-B8 861WGQGTQVTVSS 40-D7 862 WGQGTQVTVSS 40-F7 863 WGQGTQVTVSS 40-F8 864WGQGTQVTVSS 40-G7 865 WGQGTQVTVSS 40-G8 866 WGQGTQVTVSS 40-H7 867WGQGTQVTVSS x40-E8 1043 WGQGTQVTVSS

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2and CDR3 sequences present is suitably chosen from the group consistingof the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1;or from the group of CDR1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% “sequence identity” (as definedherein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” (as defined herein) with at least one ofthe CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively. More in particular, theCDR sequences are preferably chosen such that the Nanobodies of theinvention bind to a metalloproteinase from the ADAM family with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A) value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table A-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table A-1; and/or from thegroup consisting of the CDR3 sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the CDR3 sequences listed inTable A-1.

Preferably, in the Nanobodies of the invention, at least two of theCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” with at least one of the CDR1, CDR2 andCDR3 sequences, respectively, listed in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table A-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table A-1, respectively; andat least one of the CDR1 and CDR2 sequences present is suitably chosenfrom the group consisting of the CDR1 and CDR2 sequences, respectively,listed in Table A-1 or from the group of CDR1 and CDR2 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table A-1; and/or from the group consisting of the CDR1 andCDR2 sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table A-1.

Most preferably, in the Nanobodies of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1 or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least oneof the CDR1, CDR2 and CDR3 sequences present is suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table A-1. Preferably, in this aspect, at least one orpreferably both of the other two CDR sequences present are suitablychosen from CDR sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the corresponding CDR sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR sequences that have 3, 2 or only 1 amino acid difference(s) withat least one of the corresponding sequences, respectively, listed inTable A-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table A-1. Preferably, in this aspect, at least one andpreferably both of the CDR1 and CDR2 sequences present are suitablychosen from the groups of CDR1 and CDR2 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with the CDR1and CDR2 sequences, respectively, listed in Table A-1; and/or from thegroup consisting of the CDR1 and CDR2 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1and CDR2 sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table A-1. Preferably, in this aspect, the remaining CDRsequence present is suitably chosen from the group of CDR sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with at leastone of the corresponding CDR sequences listed in Table A-1; and/or fromthe group consisting of CDR sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the corresponding sequenceslisted in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the CDR3sequences listed in Table A-1, and either the CDR1 sequence or the CDR2sequence is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table A-1. Preferably, in thisaspect, the remaining CDR sequence present is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable A-1; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the corresponding CDRsequences listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, all threeCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1.

Also, generally, the combinations of CDR's listed in Table A-1 (i.e.those mentioned on the same line in Table A-1) are preferred. Thus, itis generally preferred that, when a CDR in a Nanobody of the inventionis a CDR sequence mentioned in Table A-1 or is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table A-1; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table A-1, that at least oneand preferably both of the other CDR's are suitably chosen from the CDRsequences that belong to the same combination in Table A-1 (i.e.mentioned on the same line in Table A-1) or are suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDR's mentioned in TableA-1.

Thus, by means of non-limiting examples, a Nanobody of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table A-1, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table A-1 (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or1 amino acid difference with one of the CDR2 sequences mentioned inTable A-1 (but belonging to a different combination); and a CDR3sequence that has more than 80% sequence identity with one of the CDR3sequences mentioned in Table A-1 (but belonging to a differentcombination); or (2) a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table A-1; a CDR2sequence, and one of the CDR3 sequences listed in Table A-1; or (3) aCDR1 sequence; a CDR2 sequence that has more than 80% sequence identitywith one of the CDR2 sequence listed in Table A-1; and a CDR3 sequencethat has 3, 2 or 1 amino acid differences with the CDR3 sequencementioned in Table A-1 that belongs to the same combination as the CDR2sequence.

Some particularly preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequencethat has 3, 2 or 1 amino acid difference with the CDR2 sequencementioned in Table A-1 that belongs to the same combination; and a CDR3sequence that has more than 80% sequence identity with the CDR3 sequencementioned in Table A-1 that belongs to the same combination; (2) a CDR1sequence; a CDR 2 listed in Table A-1 and a CDR3 sequence listed inTable A-1 (in which the CDR2 sequence and CDR3 sequence may belong todifferent combinations).

Some even more preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table A-1; the CDR2 sequencelisted in Table A-1 that belongs to the same combination; and a CDR3sequence mentioned in Table A-1 that belongs to a different combination;or (2) a CDR1 sequence mentioned in Table A-1; a CDR2 sequence that has3, 2 or 1 amino acid differences with the CDR2 sequence mentioned inTable A-1 that belongs to the same combination; and a CDR3 sequence thathas more than 80% sequence identity with the CDR3 sequence listed inTable A-1 that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for examplecomprise a CDR1 sequence mentioned in Table A-1, a CDR2 sequence thathas more than 80% sequence identity with the CDR2 sequence mentioned inTable A-1 that belongs to the same combination; and the CDR3 sequencementioned in Table A-1 that belongs to the same combination.

In the most preferred Nanobodies of the invention, the CDR1, CDR2 andCDR3 sequences present are suitably chosen from one of the combinationsof CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences (as defined herein) have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 868 to 973and/or 1044 to 1053.

Generally, Nanobodies with the above CDR sequences may be as furtherdescribed herein, and preferably have framework sequences that are alsoas further described herein. Thus, for example and as mentioned herein,such Nanobodies may be naturally occurring

Nanobodies (from any suitable species), naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences or Nanobodies, including but notlimited to partially humanized Nanobodies or V_(HH) sequences, fullyhumanized Nanobodies or V_(HH) sequences, camelized heavy chain variabledomain sequences, as well as Nanobodies that have been obtained by thetechniques mentioned herein.

Thus, in one specific, but non-limiting aspect, the invention relates toa humanized Nanobody, which consists of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 to CDR3 are as defined herein and in whichsaid humanized Nanobody comprises at least one humanizing substitution(as defined herein), and in particular at least one humanizingsubstitution in at least one of its framework sequences (as definedherein).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 868 to 973and/or 1044 to 1053. This degree of amino acid identity can for examplebe determined by determining the degree of amino acid identity (in amanner described herein) between said Nanobody and one or more of thesequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053, in which theamino acid residues that form the framework regions are disregarded.Such Nanobodies can be as further described herein.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody with an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: 868 to 973 and/or 1044 to 1053 or from thegroup consisting of from amino acid sequences that have more than 80%,preferably more than 90%, more preferably more than 95%, such as 99% ormore sequence identity (as defined herein) with at least one of theamino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053.

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the Nanobodies of SEQ ID NO's: 868 to 973 and/or1044 to 1053, that comprise, compared to the corresponding native V_(HH)sequence, at least one humanizing substitution (as defined herein), andin particular at least one humanizing substitution in at least one ofits framework sequences (as defined herein).

The polypeptides of the invention comprise or essentially consist of atleast one Nanobody of the invention.

It will be clear to the skilled person that the Nanobodies that arementioned herein as “preferred” (or “more preferred”, “even morepreferred”, etc.) are also preferred (or more preferred, or even morepreferred, etc.) for use in the polypeptides described herein. Thus,polypeptides that comprise or essentially consist of one or more“preferred” Nanobodies of the invention will generally be preferred, andpolypeptides that comprise or essentially consist of one or more “morepreferred” Nanobodies of the invention will generally be more preferred,etc.

Generally, proteins or polypeptides that comprise or essentially consistof a single Nanobody (such as a single Nanobody of the invention) willbe referred to herein as “monovalent” proteins or polypeptides or as“monovalent constructs”. Proteins and polypeptides that comprise oressentially consist of two or more Nanobodies (such as at least twoNanobodies of the invention or at least one Nanobody of the inventionand at least one other Nanobody) will be referred to herein as“multivalent” proteins or polypeptides or as “multivalent constructs”,and these may provide certain advantages compared to the correspondingmonovalent Nanobodies of the invention. Some non-limiting examples ofsuch multivalent constructs will become clear from the furtherdescription herein.

According to one specific, but non-limiting aspect, a polypeptide of theinvention comprises or essentially consists of at least two Nanobodiesof the invention, such as two or three Nanobodies of the invention. Asfurther described herein, such multivalent constructs can providecertain advantages compared to a protein or polypeptide comprising oressentially consisting of a single Nanobody of the invention, such as amuch improved avidity for a metalloproteinase from the ADAM family. Suchmultivalent constructs will be clear to the skilled person based on thedisclosure herein.

According to another specific, but non-limiting aspect, a polypeptide ofthe invention comprises or essentially consists of at least one Nanobodyof the invention and at least one other binding unit (i.e. directedagainst another epitope, antigen, target, protein or polypeptide), whichis preferably also a Nanobody. Such proteins or polypeptides are alsoreferred to herein as “multispecific” proteins or polypeptides or as‘multispecific constructs“, and these may provide certain advantagescompared to the corresponding monovalent Nanobodies of the invention (aswill become clear from the further discussion herein of some preferred,but-nonlimiting multispecific constructs). Such multispecific constructswill be clear to the skilled person based on the disclosure herein.

According to yet another specific, but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one Nanobody of the invention, optionally one or more furtherNanobodies, and at least one other amino acid sequence (such as aprotein or polypeptide) that confers at least one desired property tothe Nanobody of the invention and/or to the resulting fusion protein.Again, such fusion proteins may provide certain advantages compared tothe corresponding monovalent Nanobodies of the invention. Somenon-limiting examples of such amino acid sequences and of such fusionconstructs will become clear from the further description herein.

It is also possible to combine two or more of the above aspects, forexample to provide a trivalent bispecific construct comprising twoNanobodies of the invention and one other Nanobody, and optionally oneor more other amino acid sequences. Further non-limiting examples ofsuch constructs, as well as some constructs that are particularlypreferred within the context of the present invention, will become clearfrom the further description herein.

In the above constructs, the one or more Nanobodies and/or other aminoacid sequences may be directly linked to each other and/or suitablylinked to each other via one or more linker sequences. Some suitable butnon-limiting examples of such linkers will become clear from the furtherdescription herein.

In one specific aspect of the invention, a Nanobody of the invention ora compound, construct or polypeptide of the invention comprising atleast one Nanobody of the invention may have an increased half-life,compared to the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such Nanobodies, compounds andpolypeptides will become clear to the skilled person based on thefurther disclosure herein, and for example comprise Nanobodies sequencesor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);amino acid sequences of the invention that comprise at least oneadditional binding site for binding to a serum protein (such as serumalbumin, see for example EP 0 368 684 B1, page 4); or polypeptides ofthe invention that comprise at least one Nanobody of the invention thatis linked to at least one moiety (and in particular at least one aminoacid sequence) that increases the half-life of the Nanobody of theinvention. Examples of polypeptides of the invention that comprise suchhalf-life extending moieties or amino acid sequences will become clearto the skilled person based on the further disclosure herein; and forexample include, without limitation, polypeptides in which the one ormore Nanobodies of the invention are suitable linked to one or moreserum proteins or fragments thereof (such as serum albumin or suitablefragments thereof) or to one or more binding units that can bind toserum proteins (such as, for example, Nanobodies or (single) domainantibodies that can bind to serum proteins such as serum albumin, serumimmunoglobulins such as IgG, or transferrin); polypeptides in which aNanobody of the invention is linked to an Fc portion (such as a humanFc) or a suitable part or fragment thereof; or polypeptides in which theone or more Nanobodies of the invention are suitable linked to one ormore small proteins or peptides that can bind to serum proteins (suchas, without limitation, the proteins and peptides described in WO91/01743, WO 01/45746, WO 02/076489 and to the US provisionalapplication of Ablynx N.V. entitled “Peptides capable of binding toserum proteins” of Ablynx N. V. filed on Dec. 5, 2006 (see alsoPCT/EP/2007/063348).

Again, as will be clear to the skilled person, such Nanobodies,compounds, constructs or polypeptides may contain one or more additionalgroups, residues, moieties or binding units, such as one or more furtheramino acid sequences and in particular one or more additional Nanobodies(i.e. not directed against a metalloproteinase from the ADAM family), soas to provide a tri- of multispecific Nanobody construct.

Generally, the Nanobodies of the invention (or compounds, constructs orpolypeptides comprising the same) with increased half-life preferablyhave a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, for example at least 10 times or morethan 20 times, greater than the half-life of the corresponding aminoacid sequence of the invention per se. For example, the Nanobodies,compounds, constructs or polypeptides of the invention with increasedhalf-life may have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchNanobodies, compound, constructs or polypeptides of the inventionexhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), preferably at least 9 days (such as about9 to 14 days), more preferably at least about 10 days (such as about 10to 15 days), or at least about 11 days (such as about 11 to 16 days),more preferably at least about 12 days (such as about 12 to 18 days ormore), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the inventioncomprises one or more (such as two or preferably one) Nanobodies of theinvention linked (optionally via one or more suitable linker sequences)to one or more (such as two and preferably one) amino acid sequencesthat allow the resulting polypeptide of the invention to cross the bloodbrain barrier. In particular, said one or more amino acid sequences thatallow the resulting polypeptides of the invention to cross the bloodbrain barrier may be one or more (such as two and preferably one)Nanobodies, such as the Nanobodies described in WO 02/057445, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In particular, polypeptides comprising one or more Nanobodies of theinvention are preferably such that they:

-   -   bind to a metalloproteinase from the ADAM family with a        dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or        less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more        preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association        constant (K_(A)) of 10⁵ to 10¹² liter/ moles or more, and        preferably 10⁷ to 10¹² liter/moles or more and more preferably        10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to a metalloproteinase from the ADAM family with a        k_(on)-rate of between 10²M⁻¹ s⁻¹ to about 10⁷M⁻¹ s⁻¹,        preferably between 10³M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, more preferably        between 10⁴M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, such as between 10⁵M⁻¹s⁻¹ and        10 ⁷M⁻¹ s⁻¹;        and/or such that they:    -   bind to a metalloproteinase from the ADAM family with a k_(off)        rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a        near irreversible complex with a t_(1/2) of multiple days),        preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably        between 10⁻³ s⁻¹ and 10⁻⁴ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶        s⁻¹.

Preferably, a polypeptide that contains only one amino acid sequence ofthe invention is preferably such that it will bind to ametalloproteinase from the ADAM family with an affinity less than 500nM, preferably less than 200 nM, more preferably less than 10 nM, suchas less than 500 pM. In this respect, it will be clear to the skilledperson that a polypeptide that contains two or more Nanobodies of theinvention may bind to a metalloproteinase from the ADAM family with anincreased avidity, compared to a polypeptide that contains only oneamino acid sequence of the invention.

Some preferred IC₅₀ values for binding of the amino acid sequences orpolypeptides of the invention to a metalloproteinase from the ADAMfamily will become clear from the further description and examplesherein.

Another aspect of this invention relates to a nucleic acid that encodesan amino acid sequence of the invention (such as a Nanobody of theinvention) or a polypeptide of the invention comprising the same. Again,as generally described herein for the nucleic acids of the invention,such a nucleic acid may be in the form of a genetic construct, asdefined herein.

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing an amino acid sequence (suchas a Nanobody) of the invention and/or a polypeptide of the inventioncomprising the same; and/or that contains a nucleic acid of theinvention. Some preferred but non-limiting examples of such hosts orhost cells will become clear from the further description herein.

Another aspect of the invention relates to a product or compositioncontaining or comprising at least one amino acid sequence of theinvention, at least one polypeptide of the invention and/or at least onenucleic acid of the invention, and optionally one or more furthercomponents of such compositions known per se, i.e. depending on theintended use of the composition. Such a product or composition may forexample be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention further relates to methods for preparing or generating theamino acid sequences, compounds, constructs, polypeptides, nucleicacids, host cells, products and compositions described herein. Somepreferred but non-limiting examples of such methods will become clearfrom the further description herein.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with a metalloproteinase from the ADAM family. Some preferredbut non-limiting applications and uses will become clear from thefurther description herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description hereinbelow.

Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can generally beobtained: (1) by isolating the V_(HH) domain of a naturally occurringheavy chain antibody; (2) by expression of a nucleotide sequenceencoding a naturally occurring V_(HH) domain; (3) by “humanization” (asdescribed herein) of a naturally occurring V_(HH) domain or byexpression of a nucleic acid encoding a such humanized V_(HH) domain;(4) by “camelization” (as described herein) of a naturally occurringV_(H) domain from any animal species, and in particular a from speciesof mammal, such as from a human being, or by expression of a nucleicacid encoding such a camelized V_(H) domain; (5) by “camelisation” of a“domain antibody” or “Dab” as described by Ward et al (supra), or byexpression of a nucleic acid encoding such a camelized V_(H) domain; (6)by using synthetic or semi-synthetic techniques for preparing proteins,polypeptides or other amino acid sequences known per se; (7) bypreparing a nucleic acid encoding a Nanobody using techniques fornucleic acid synthesis known per se, followed by expression of thenucleic acid thus obtained; and/or (8) by any combination of one or moreof the foregoing. Suitable methods and techniques for performing theforegoing will be clear to the skilled person based on the disclosureherein and for example include the methods and techniques described inmore detail herein.

One preferred class of Nanobodies corresponds to the V_(HH) domains ofnaturally occurring heavy chain antibodies directed against ametalloproteinase from the ADAM family. As further described herein,such V_(HH) sequences can generally be generated or obtained by suitablyimmunizing a species of Camelid with a metalloproteinase from the ADAMfamily (i.e. so as to raise an immune response and/or heavy chainantibodies directed against a metalloproteinase from the ADAM family),by obtaining a suitable biological sample from said Camelid (such as ablood sample, serum sample or sample of B-cells), and by generatingV_(HH) sequences directed against a metalloproteinase from the ADAMfamily, starting from said sample, using any suitable technique knownper se. Such techniques will be clear to the skilled person and/or arefurther described herein.

Alternatively, such naturally occurring V_(HH) domains against ametalloproteinase from the ADAM family, can be obtained from naïvelibraries of Camelid V_(HH) sequences, for example by screening such alibrary using a metalloproteinase from the ADAM family, or at least onepart, fragment, antigenic determinant or epitope thereof using one ormore screening techniques known per se. Such libraries and techniquesare for example described in WO 99/37681, WO 01/90190, WO 03/025020 andWO 03/035694. Alternatively, improved synthetic or semi-syntheticlibraries derived from naïve V_(HH) libraries may be used, such asV_(HH) libraries obtained from naïve V_(HH) libraries by techniques suchas random mutagenesis and/or CDR shuffling, as for example described inWO 00/43507.

Thus, in another aspect, the invention relates to a method forgenerating Nanobodies, that are directed against a metalloproteinasefrom the ADAM family. In one aspect, said method at least comprises thesteps of:

-   -   a) providing a set, collection or library of Nanobody sequences;        and    -   b) screening said set, collection or library of Nanobody        sequences for Nanobody sequences that can bind to and/or have        affinity for a metalloproteinase from the ADAM family;        and    -   c) isolating the amino acid sequence(s) that can bind to and/or        have affinity for a metalloproteinase from the ADAM family.

In such a method, the set, collection or library of Nanobody sequencesmay be a naïve set, collection or library of Nanobody sequences; asynthetic or semi-synthetic set, collection or library of Nanobodysequences; and/or a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofNanobody sequences may be an immune set, collection or library ofNanobody sequences, and in particular an immune set, collection orlibrary of V_(HH) sequences, that have been derived from a species ofCamelid that has been suitably immunized with a metalloproteinase fromthe ADAM family or with a suitable antigenic determinant based thereonor derived therefrom, such as an antigenic part, fragment, region,domain, loop or other epitope thereof. In one particular aspect, saidantigenic determinant may be an extracellular part, region, domain, loopor other extracellular epitope(s).

In the above methods, the set, collection or library of Nanobody orV_(HH) sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) Nanobodysequences will be clear to the person skilled in the art, for example onthe basis of the further disclosure herein. Reference is also made to WO03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23,9, 1105-1116 (2005).

In another aspect, the method for generating Nanobody sequencescomprises at least the steps of:

-   -   a) providing a collection or sample of cells derived from a        species of Camelid that express immunoglobulin sequences;    -   b) screening said collection or sample of cells for (i) cells        that express an immunoglobulin sequence that can bind to and/or        have affinity for a metalloproteinase from the ADAM family;        and (ii) cells that express heavy chain antibodies, in which        substeps (i) and (ii) can be performed essentially as a single        screening step or in any suitable order as two separate        screening steps, so as to provide at least one cell that        expresses a heavy chain antibody that can bind to and/or has        affinity for a metalloproteinase from the ADAM family;        and    -   c) either (i) isolating from said cell the Vim sequence present        in said heavy chain antibody; or (ii) isolating from said cell a        nucleic acid sequence that encodes the V_(HH) sequence present        in said heavy chain antibody, followed by expressing said V_(HH)        domain.

In the method according to this aspect, the collection or sample ofcells may for example be a collection or sample of B-cells. Also, inthis method, the sample of cells may be derived from a Camelid that hasbeen suitably immunized with a metalloproteinase from the ADAM family ora suitable antigenic determinant based thereon or derived therefrom,such as an antigenic part, fragment, region, domain, loop or otherepitope thereof. In one particular aspect, said antigenic determinantmay be an extracellular part, region, domain, loop or otherextracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820. Particular reference is made to the so-called“Nanoclone™” technique described in International application WO06/079372 by Ablynx N. V.

In another aspect, the method for generating an amino acid sequencedirected against a metalloproteinase from the ADAM family may compriseat least the steps of:

-   -   a) providing a set, collection or library of nucleic acid        sequences encoding heavy chain antibodies or Nanobody sequences;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode a heavy chain        antibody or a Nanobody sequence that can bind to and/or has        affinity for a metalloproteinase from the ADAM family;        and    -   c) isolating said nucleic acid sequence, followed by expressing        the V_(HH) sequence present in said heavy chain antibody or by        expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding heavy chain antibodies or Nanobody sequences may forexample be a set, collection or library of nucleic acid sequencesencoding a naïve set, collection or library of heavy chain antibodies orV_(HH) sequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofNanobody sequences; and/or a set, collection or library of nucleic acidsequences encoding a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences encoding heavy chain antibodies or V_(HH)sequences derived from a Camelid that has been suitably immunized with ametalloproteinase from the ADAM family or with a suitable antigenicdeterminant based thereon or derived therefrom, such as an antigenicpart, fragment, region, domain, loop or other epitope thereof. In oneparticular aspect, said antigenic determinant may be an extracellularpart, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to WO 03/054016 and to the review by Hoogenboomin Nature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of themethods described herein can also be performed as a selection step.Accordingly the term “screening” as used in the present description cancomprise selection, screening or any suitable combination of selectionand/or screening techniques. Also, when a set, collection or library ofsequences is used, it may contain any suitable number of sequences, suchas 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collectionor library of amino acid sequences may be obtained or defined byrational, or semi-empirical approaches such as computer modellingtechniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two ormore sequences that are variants from one another (e.g. with designedpoint mutations or with randomized positions), compromise multiplesequences derived from a diverse set of naturally diversified sequences(e.g. an immune library)), or any other source of diverse sequences (asdescribed for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection orlibrary of sequences can be displayed on the surface of a phageparticle, a ribosome, a bacterium, a yeast cell, a mammalian cell, andlinked to the nucleotide sequence encoding the amino acid sequencewithin these carriers. This makes such set, collection or libraryamenable to selection procedures to isolate the desired amino acidsequences of the invention. More generally, when a sequence is displayedon a suitable host or host cell, it is also possible (and customary) tofirst isolate from said host or host cell a nucleotide sequence thatencodes the desired sequence, and then to obtain the desired sequence bysuitably expressing said nucleotide sequence in a suitable hostorganism. Again, this can be performed in any suitable manner known perse, as will be clear to the skilled person.

Yet another technique for obtaining V_(HH) sequences or Nanobodysequences directed against a metalloproteinase from the ADAM familyinvolves suitably immunizing a transgenic mammal that is capable ofexpressing heavy chain antibodies (i.e. so as to raise an immuneresponse and/or heavy chain antibodies directed against ametalloproteinase from the ADAM family), obtaining a suitable biologicalsample from said transgenic mammal that contains (nucleic acid sequencesencoding) said V_(HH) sequences or Nanobody sequences (such as a bloodsample, serum sample or sample of B-cells), and then generating V_(HH)sequences directed against a metalloproteinase from the ADAM family,starting from said sample, using any suitable technique known per se(such as any of the methods described herein or a hybridoma technique).For example, for this purpose, the heavy chain antibody-expressing miceand the further methods and techniques described in WO 02/085945, WO04/049794 and WO 06/008548 and Janssens et al., Proc. Natl. Acad. Sci.USA. 2006 Oct. 10;103(41):15130-5 can be used. For example, such heavychain antibody expressing mice can express heavy chain antibodies withany suitable (single) variable domain, such as (single) variable domainsfrom natural sources (e.g. human (single) variable domains, Camelid(single) variable domains or shark (single) variable domains), as wellas for example synthetic or semi-synthetic (single) variable domains.

The invention also relates to the V_(HH) sequences or Nanobody sequencesthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid V_(HH) sequence or Nanobody sequence; and of expressing orsynthesizing said V_(HH) sequence or Nanobody sequence in a manner knownper se, such as by expression in a suitable host cell or host organismor by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of theinvention comprises Nanobodies with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been “humanized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences)by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above). This can beperformed in a manner known per se, which will be clear to the skilledperson, for example on the basis of the further description herein andthe prior art on humanization referred to herein. Again, it should benoted that such humanized Nanobodies of the invention can be obtained inany suitable manner known per se (i.e. as indicated under points (1)-(8)above) and thus are not strictly limited to polypeptides that have beenobtained using a polypeptide that comprises a naturally occurring V_(HH)domain as a starting material.

Another particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(H) domain, but that hasbeen “camelized”, i.e. by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe further description herein. Such “camelizing” substitutions arepreferably inserted at amino acid positions that form and/or are presentat the V_(H)-V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see for example WO 94/04678 and Davies andRiechmann (1994 and 1996), supra). Preferably, the V_(H) sequence thatis used as a starting material or starting point for generating ordesigning the camelized Nanobody is preferably a V_(H) sequence from amammal, more preferably the V_(H) sequence of a human being, such as aV_(H)3 sequence. However, it should be noted that such camelizedNanobodies of the invention can be obtained in any suitable manner knownper se (i.e. as indicated under points (1)-(8) above) and thus are notstrictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring V_(H) domain as astarting material.

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” Nanobody of the invention,respectively. This nucleic acid can then be expressed in a manner knownper se, so as to provide the desired Nanobody of the invention.Alternatively, based on the amino acid sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, the amino acid sequence ofthe desired humanized or camelized Nanobody of the invention,respectively, can be designed and then synthesized de novo usingtechniques for peptide synthesis known per se. Also, based on the aminoacid sequence or nucleotide sequence of a naturally occurring V_(HH)domain or V_(H) domain, respectively, a nucleotide sequence encoding thedesired humanized or camelized Nanobody of the invention, respectively,can be designed and then synthesized de novo using techniques fornucleic acid synthesis known per se, after which the nucleic acid thusobtained can be expressed in a manner known per se, so as to provide thedesired Nanobody of the invention.

Other suitable methods and techniques for obtaining the Nanobodies ofthe invention and/or nucleic acids encoding the same, starting fromnaturally occurring V_(H) sequences or preferably V_(HH) sequences, willbe clear from the skilled person, and may for example comprise combiningone or more parts of one or more naturally occurring V_(H) sequences(such as one or more FR sequences and/or CDR sequences), one or moreparts of one or more naturally occurring V_(HH) sequences (such as oneor more FR sequences or CDR sequences), and/or one or more synthetic orsemi-synthetic sequences, in a suitable manner, so as to provide aNanobody of the invention or a nucleotide sequence or nucleic acidencoding the same (which may then be suitably expressed). Nucleotidesequences encoding framework sequences of V_(HH) sequences or Nanobodieswill be clear to the skilled person based on the disclosure hereinand/or the further prior art cited herein (and/or may alternatively beobtained by PCR starting from the nucleotide sequences obtained usingthe methods described herein) and may be suitably combined withnucleotide sequences that encode the desired CDR's (for example, by PCRassembly using overlapping primers), so as to provide a nucleic acidencoding a Nanobody of the invention.

As mentioned herein, Nanobodies may in particular be characterized bythe presence of one or more “Hallmark residues” (as described herein) inone or more of the framework sequences.

Thus, according to one preferred, but non-limiting aspect of theinvention, a Nanobody in its broadest sense can be generally defined asa polypeptide comprising:

-   -   a) an amino acid sequence that is comprised of four framework        regions/sequences interrupted by three complementarity        determining regions/sequences, in which the amino acid residue        at position 108 according to the Kabat numbering is Q; and/or:    -   b) an amino acid sequence that is comprised of four framework        regions/sequences interrupted by three complementarity        determining regions/sequences, in which the amino acid residue        at position 45 according to the Kabat numbering is a charged        amino acid (as defined herein) or a cysteine residue, and        position 44 is preferably an E; and/or:    -   c) an amino acid sequence that is comprised of four framework        regions/sequences interrupted by three complementarity        determining regions/sequences, in which the amino acid residue        at position 103 according to the Kabat numbering is chosen from        the group consisting of P, R and S, and is in particular chosen        from the group consisting of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   -   a) the amino acid residue at position 108 according to the Kabat        numbering is Q; and/or in which:    -   b) the amino acid residue at position 45 according to the Kabat        numbering is a charged amino acid or a cysteine and the amino        acid residue at position 44 according to the Kabat numbering is        preferably E; and/or in which:    -   c) the amino acid residue at position 103 according to the Kabat        numbering is chosen from the group consisting of P, R and S, and        is in particular chosen from the group consisting of R and S;        and in which:    -   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably        as defined according to one of the preferred aspects herein, and        are more preferably as defined according to one of the more        preferred aspects herein.

In particular, a Nanobody in its broadest sense can be generally definedas a polypeptide comprising:

-   -   a) an amino acid sequence that is comprised of four framework        regions/sequences interrupted by three complementarity        determining regions/sequences, in which the amino acid residue        at position 108 according to the Kabat numbering is Q; and/or:    -   b) an amino acid sequence that is comprised of four framework        regions/sequences interrupted by three complementarity        determining regions/sequences, in which the amino acid residue        at position 44 according to the Kabat numbering is E and in        which the amino acid residue at position 45 according to the        Kabat numbering is an R; and/or:    -   c) an amino acid sequence that is comprised of four framework        regions/sequences interrupted by three complementarity        determining regions/sequences, in which the amino acid residue        at position 103 according to the Kabat numbering is chosen from        the group consisting of P, R and S, and is in particular chosen        from the group consisting of R and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody ofthe invention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   -   a) the amino acid residue at position 108 according to the Kabat        numbering is Q;        and/or in which:    -   b) the amino acid residue at position 44 according to the Kabat        numbering is E and in which the amino acid residue at position        45 according to the Kabat numbering is an R;        and/or in which:    -   c) the amino acid residue at position 103 according to the Kabat        numbering is chosen from the group consisting of P, R and S, and        is in particular chosen from the group consisting of R and S;        and in which:    -   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably        as defined according to one of the preferred aspects herein, and        are more preferably as defined according to one of the more        preferred aspects herein.

In particular, a Nanobody against a metalloproteinase from the ADAMfamily according to the invention may have the structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   -   a) the amino acid residue at position 108 according to the Kabat        numbering is Q; and/or in which:    -   b) the amino acid residue at position 44 according to the Kabat        numbering is E and in which the amino acid residue at position        45 according to the Kabat numbering is an R;        and/or in which:    -   c) the amino acid residue at position 103 according to the Kabat        numbering is chosen from the group consisting of P, R and S, and        is in particular chosen from the group consisting of R and S;        and in which:    -   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably        as defined according to one of the preferred aspects herein, and        are more preferably as defined according to one of the more        preferred aspects herein.

In particular, according to one preferred, but non-limiting aspect ofthe invention, a Nanobody can generally be defined as a polypeptidecomprising an amino acid sequence that is comprised of four frameworkregions/sequences interrupted by three complementarity determiningregions/sequences, in which;

-   -   a-1) the amino acid residue at position 44 according to the        Kabat numbering is chosen from the group consisting of A, G, E,        D, G, Q, R, S, L; and is preferably chosen from the group        consisting of G, E or Q; and    -   a-2) the amino acid residue at position 45 according to the        Kabat numbering is chosen from the group consisting of L, R or        C; and is preferably chosen from the group consisting of L or R;        and    -   a-3) the amino acid residue at position 103 according to the        Kabat numbering is chosen from the group consisting of W, R or        S; and is preferably W or R, and is most preferably W;    -   a-4) the amino acid residue at position 108 according to the        Kabat numbering is Q;        or in which:    -   b-1) the amino acid residue at position 44 according to the        Kabat numbering is chosen from the group consisting of E and Q;        and    -   b-2) the amino acid residue at position 45 according to the        Kabat numbering is R; and    -   b-3) the amino acid residue at position 103 according to the        Kabat numbering is chosen from the group consisting of W, R and        S; and is preferably W;    -   b-4) the amino acid residue at position 108 according to the        Kabat numbering is chosen from the group consisting of Q and L;        and is preferably Q;        or in which:    -   c-1) the amino acid residue at position 44 according to the        Kabat numbering is chosen from the group consisting of A, G, E,        D, Q, R, S and L; and is preferably chosen from the group        consisting of G, E and Q; and    -   c-2) the amino acid residue at position 45 according to the        Kabat numbering is chosen from the group consisting of L, R and        C; and is preferably chosen from the group consisting of L and        R; and    -   c-3) the amino acid residue at position 103 according to the        Kabat numbering is chosen from the group consisting of P, R and        S; and is in particular chosen from the group consisting of R        and S; and    -   c-4) the amino acid residue at position 108 according to the        Kabat numbering is chosen from the group consisting of Q and L;        is preferably Q;        and in which    -   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably        as defined according to one of the preferred aspects herein, and        are more preferably as defined according to one of the more        preferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   -   a-1) the amino acid residue at position 44 according to the        Kabat numbering is chosen from the group consisting of A, G, E,        D, G, Q, R, S, L; and is preferably chosen from the group        consisting of G, E or Q;        and in which:    -   a-2) the amino acid residue at position 45 according to the        Kabat numbering is chosen from the group consisting of L, R or        C; and is preferably chosen from the group consisting of L or R;        and in which:    -   a-3) the amino acid residue at position 103 according to the        Kabat numbering is chosen from the group consisting of W, R or        S; and is preferably W or R, and is most preferably W;        and in which    -   a-4) the amino acid residue at position 108 according to the        Kabat numbering is Q;        and in which:    -   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably        as defined according to one of the preferred aspects herein, and        are more preferably as defined according to one of the more        preferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   -   b-1) the amino acid residue at position 44 according to the        Kabat numbering is chosen from the group consisting of E and Q;        and in which:    -   b-2) the amino acid residue at position 45 according to the        Kabat numbering is R;        and in which:    -   b-3) the amino acid residue at position 103 according to the        Kabat numbering is chosen from the group consisting of W, R and        S; and is preferably W;        and in which:    -   b-4) the amino acid residue at position 108 according to the        Kabat numbering is chosen from the group consisting of Q and L;        and is preferably Q;        and in which:    -   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably        as defined according to one of the preferred aspects herein, and        are more preferably as defined according to one of the more        preferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   -   c-1) the amino acid residue at position 44 according to the        Kabat numbering is chosen from the group consisting of A, G, E,        D, Q, R, S and L; and is preferably chosen from the group        consisting of G, E and Q;        and in which:    -   c-2) the amino acid residue at position 45 according to the        Kabat numbering is chosen from the group consisting of L, R and        C; and is preferably chosen from the group consisting of L and        R;        and in which:    -   c-3) the amino acid residue at position 103 according to the        Kabat numbering is chosen from the group consisting of P, R and        S; and is in particular chosen from the group consisting of R        and S;        and in which:    -   c-4) the amino acid residue at position 108 according to the        Kabat numbering is chosen from the group consisting of Q and L;        is preferably Q;        and in which:    -   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably        as defined according to one of the preferred aspects herein, and        are more preferably as defined according to one of the more        preferred aspects herein.

Two particularly preferred, but non-limiting groups of the Nanobodies ofthe invention are those according to a) above; according to (a-1) to(a-4) above; according to b) above; according to (b-1) to (b-4) above;according to (c) above; and/or according to (c-1) to (c-4) above, inwhich either:

-   -   i) the amino acid residues at positions 44-47 according to the        Kabat numbering form the sequence GLEW (or a GLEW-like sequence        as described herein) and the amino acid residue at position 108        is Q;        or in which:    -   ii) the amino acid residues at positions 43-46 according to the        Kabat numbering form the sequence KERE or KQRE (or a KERE-like        sequence as described) and the amino acid residue at position        108 is Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   -   i) the amino acid residues at positions 44-47 according to the        Kabat numbering form the sequence GLEW (or a GLEW-like sequence        as defined herein) and the amino acid residue at position 108 is        Q;        and in which:    -   ii) CDR1, CDR2 and CDR3 are as defined herein, and are        preferably as defined according to one of the preferred aspects        herein, and are more preferably as defined according to one of        the more preferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   -   i) the amino acid residues at positions 43-46 according to the        Kabat numbering form the sequence KERE or KQRE (or a KERE-like        sequence) and the amino acid residue at position 108 is Q or L,        and is preferably Q;        and in which:    -   ii) CDR1, CDR2 and CDR3 are as defined herein, and are        preferably as defined according to one of the preferred aspects        herein, and are more preferably as defined according to one of        the more preferred aspects herein.

In the Nanobodies of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KEREor KQRE, the amino acid residue at position 37 is most preferably F. Inthe Nanobodies of the invention in which the amino acid residues atpositions 44-47 according to the Kabat numbering form the sequence GLEW,the amino acid residue at position 37 is chosen from the groupconsisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the Nanobodiesof the invention can generally be classified on the basis of thefollowing three groups:

-   -   i) The “GLEW-group”: Nanobodies with the amino acid sequence        GLEW at positions 44-47 according to the Kabat numbering and Q        at position 108 according to the Kabat numbering. As further        described herein, Nanobodies within this group usually have a V        at position 37, and can have a W, P, R or S at position 103, and        preferably have a W at position 103. The GLEW group also        comprises some GLEW-like sequences such as those mentioned in        Table A-3 below. More generally, and without limitation,        Nanobodies belonging to the GLEW-group can be defined as        Nanobodies with a G at position 44 and/or with a W at position        47, in which position 46 is usually E and in which preferably        position 45 is not a charged amino acid residue and not        cysteine;    -   ii) The “KERE-group”: Nanobodies with the amino acid sequence        KERE or KQRE (or another KERE-like sequence) at positions 43-46        according to the Kabat numbering and Q or L at position 108        according to the Kabat numbering. As further described herein,        Nanobodies within this group usually have a F at position 37, an        L or F at position 47; and can have a W, P, R or S at position        103, and preferably have a W at position 103. More generally,        and without limitation, Nanobodies belonging to the KERE-group        can be defined as Nanobodies with a K, Q or R at position 44        (usually K) in which position 45 is a charged amino acid residue        or cysteine, and position 47 is as further defined herein;    -   iii) The “103 P, R, S-group”: Nanobodies with a P, R or S at        position 103. These Nanobodies can have either the amino acid        sequence GLEW at positions 44-47 according to the Kabat        numbering or the amino acid sequence KERE or KQRE at positions        43-46 according to the Kabat numbering, the latter most        preferably in combination with an F at position 37 and an L or        an F at position 47 (as defined for the KERE-group); and can        have Q or L at position 108 according to the Kabat numbering,        and preferably have Q.

Also, where appropriate, Nanobodies may belong to (i.e. havecharacteristics of) two or more of these classes. For example, onespecifically preferred group of Nanobodies has GLEW or a GLEW-likesequence at positions 44-47; P, R or S (and in particular R) at position103; and Q at position 108 (which may be humanized to L). Moregenerally, it should be noted that the definitions referred to abovedescribe and apply to Nanobodies in the form of a native (i.e.non-humanized) V_(HH) sequence, and that humanized variants of theseNanobodies may contain other amino acid residues than those indicatedabove (i.e. one or more humanizing substitutions as defined herein). Forexample, and without limitation, in some humanized Nanobodies of theGLEW-group or the 103 P, R, S-group, Q at position 108 may be humanizedto 108L. As already mentioned herein, other humanizing substitutions(and suitable combinations thereof) will become clear to the skilledperson based on the disclosure herein. In addition, or alternatively,other potentially useful humanizing substitutions can be ascertained bycomparing the sequence of the framework regions of a naturally occurringV_(HH) sequence with the corresponding framework sequence of one or moreclosely related human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence (in anymanner known per se, as further described herein) and the resultinghumanized V_(HH) sequences can be tested for affinity for the target,for stability, for ease and level of expression, and/or for otherdesired properties. In this way, by means of a limited degree of trialand error, other suitable humanizing substitutions (or suitablecombinations thereof) can be determined by the skilled person based onthe disclosure herein. Also, based on the foregoing, (the frameworkregions of) a Nanobody may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the KERE-group (as definedherein), and CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the 103 P, R, S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined herein,and are preferably as defined according to one of the preferred aspectsherein, and are more preferably as defined according to one of the morepreferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P,R,Sresidues mentioned above, the Nanobodies of the invention can contain,at one or more positions that in a conventional V_(H) domain would form(part of) the V_(H)/V_(L) interface, one or more amino acid residuesthat are more highly charged than the amino acid residues that naturallyoccur at the same position(s) in the corresponding naturally occurringV_(H) sequence, and in particular one or more charged amino acidresidues (as mentioned in Table A-2). Such substitutions include, butare not limited to, the GLEW-like sequences mentioned in Table A-3below; as well as the substitutions that are described in theInternational Application WO 00/29004 for so-called “microbodies”, e.g.so as to obtain a Nanobody with Q at position 108 in combination withKLEW at positions 44-47. Other possible substitutions at these positionswill be clear to the skilled person based upon the disclosure herein.

In one aspect of the Nanobodies of the invention, the amino acid residueat position 83 is chosen from the group consisting of L, M, S, V and W;and is preferably L.

Also, in one aspect of the Nanobodies of the invention, the amino acidresidue at position 83 is chosen from the group consisting of R, K, N,E, G, I, T and Q; and is most preferably either K or E (for Nanobodiescorresponding to naturally occurring V_(HH) domains) or R (for“humanized” Nanobodies, as described herein). The amino acid residue atposition 84 is chosen from the group consisting of P, A, R, S, D T, andV in one aspect, and is most preferably P (for Nanobodies correspondingto naturally occurring V_(HH) domains) or R (for “humanized” Nanobodies,as described herein).

Furthermore, in one aspect of the Nanobodies of the invention, the aminoacid residue at position 104 is chosen from the group consisting of Gand D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the Nanobodies are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human V_(H) domain, V_(H)3, aresummarized in Table A-3.

Some especially preferred but non-limiting combinations of theseHallmark Residues as occur in naturally occurring V_(HH) domains arementioned in Table A-4. For comparison, the corresponding amino acidresidues of the human V_(H)3 called DP-47 have been indicated initalics.

TABLE A-3 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues  11 L, V; predominantly L L, M, S, V, W; preferably L  37 V, I,F; usually V F⁽¹⁾, Y, H, I, L or V, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G G⁽²⁾,E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾or E⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, V or Y; preferablyW⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R  83 R or K; usually R R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Qor T; preferably K or R; most preferably K  84 A, T, D; predominantly AP⁽⁵⁾, A, L, R, S, T, D, V; preferably P 103 W W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S;preferably W 104 G G or D; preferably G 108 L, M or T; predominantly LQ, L⁽⁷⁾ or R; preferably Q or L⁽⁷⁾ Notes: ⁽¹⁾In particular, but notexclusively, in combination with KERE or KQRE at positions 43-46.⁽²⁾Usually as GLEW at positions 44-47. ⁽³⁾Usually as KERE or KQRE atpositions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF or KEREG atpositions 43-47. Alternatively, also sequences such as TERE (for exampleTEREL), KECE (for example KECEL or KECER), RERE (for example REREG),QERE (for example QEREG), KGRE (for example KGREG), KDRE (for exampleKDREV) are possible. Some other possible, but less preferred sequencesinclude for example DECKL and NVCEL. ⁽⁴⁾With both GLEW at positions44-47 and KERE or KQRE at positions 43-46. ⁽⁵⁾Often as KP or EP atpositions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾In particular,but not exclusively, in combination with GLEW at positions 44-47.⁽⁷⁾With the proviso that when positions 44-47 are GLEW, position 108 isalways Q in (non-humanized) V_(HH) sequences that also contain a W atposition 103. The GLEW group also contains GLEW-like sequences atpositions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW,ELEW, GPEW, EWLP, GPER, GLER and ELEW.

TABLE A-4 Some preferred but non-limiting combinations of HallmarkResidues in naturally occurring Nanobodies. 11 37 44 45 47 83 84 103 104108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R L K P W G Q LF E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q L F L R V K PQ G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group L V G L W K SW G Q M V G L W K P R G Q For humanization of these combinations,reference is made to the specification.

In the Nanobodies, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables A-5to A-8 mention some non-limiting residues that can be present at eachposition (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4of naturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a Nanobody) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering by Chothia (supra) that the residues at thesepositions already form part of CDR1.)

In Tables A-5-A-8, some of the non-limiting residues that can be presentat each position of a human V_(H)3 domain have also been mentioned.Again, for each position, the amino acid residue that most frequentlyoccurs at each position of a naturally occurring human V_(H)3 domain isindicated in bold; and other preferred amino acid residues have beenunderlined.

For reference only, Tables A-5-A-8 also contain data on the V_(HH)entropy (“V_(HH) Ent.”) and V_(HH) variability (“V_(HH) Var.”) at eachamino acid position for a representative sample of 1118 V_(HH) sequences(data kindly provided by David Lutje Hulsing and Prof. Theo Verrips ofUtrecht University). The values for the V_(HH) entropy and the V_(HH)variability provide a measure for the variability and degree ofconservation of amino acid residues between the 1118 V_(HH) sequencesanalyzed: low values (i.e. <1, such as <0.5) indicate that an amino acidresidue is highly conserved between the V_(HH) sequences (i.e. littlevariability). For example, the G at position 8 and the G at position 9have values for the V_(HH) entropy of 0.1 and 0 respectively, indicatingthat these residues are highly conserved and have little variability(and in case of position 9 is G in all 1118 sequences analysed), whereasfor residues that form part of the CDR's generally values of 1.5 or moreare found (data not shown). Note that (1) the amino acid residues listedin the second column of Tables A-5-A-8 are based on a bigger sample thanthe 1118 V_(HH) sequences that were analysed for determining the V_(HH)entropy and V_(HH) variability referred to in the last two columns; and(2) the data represented below support the hypothesis that the aminoacid residues at positions 27-30 and maybe even also at positions 93 and94 already form part of the CDR's (although the invention is not limitedto any specific hypothesis or explanation, and as mentioned above,herein the numbering according to Kabat is used). For a generalexplanation of sequence entropy, sequence variability and themethodology for determining the same, see Oliveira et al., PROTEINS:Structure, Function and Genetics, 52: 544-552 (2003).

TABLE A-5 Non-limiting examples of amino acid residues in FR1 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 1 E, Q Q, A, E — — 2V V 0.2 1 3 Q Q, K 0.3 2 4 L L 0.1 1 5 V, L Q, E, L, V 0.8 3 6 E E, D,Q, A 0.8 4 7 S, T S, F 0.3 2 8 G, R G 0.1 1 9 G G 0 1 10 G, V G, D, R0.3 2 11 Hallmark residue: L, M, S, V, W; preferably L 0.8 2 12 V, I V,A 0.2 2 13 Q, K, R Q, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1 515 G G 0 1 16 G, R G, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1 1 19R, K R, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A, F, T 0.2 322 C C 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I, L, S, T, V 1 625 S S, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V 0.7 7 27 F S, F, R,L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A, G, R, F, Y 1.7 11 29 F,V F, L, D, S, I, G, V, A 1.9 11 30 S, D, G N, S, E, G, A, D, M, T 1.8 11

TABLE A-6 Non-limiting examples of amino acid residues in FR2 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 36 W W 0.1 1 37Hallmark residue: F⁽¹⁾, H, I, L, Y or V, preferably F⁽¹⁾ or Y 1.1 6 38 RR 0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S, TP, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7 6 44Hallmark residue: G⁽²⁾, E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or1.3 5 Q; most preferably G⁽²⁾ or E⁽³⁾ 45 Hallmark residue: L⁽²⁾, R⁽³⁾,C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 0.6 4 46 E, V E, D, K, Q, V0.4 2 47 Hallmark residue: W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, V or Y;1.9 9 preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L 0.4 3 49 S, A, G A,S, G, T, V 0.8 3

TABLE A-7 Non-limiting examples of amino acid residues in FR3 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 66 R R 0.1 1 67 F F,L, V 0.1 1 68 T T, A, N, S 0.5 4 69 I I, L, M, V 0.4 4 70 S S, A, F, T0.3 4 71 R R, G, H, I, L, K, Q, S, T, W 1.2 8 72 D, E D, E, G, N, V 0.54 73 N, D, G N, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G,N, P, S, T, V 1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D, K, R,S, T, Y 0.9 6 77 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V, L, A, F,G, I, M 1.2 5 79 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L, F, V 0.1 1 81 QQ, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2  82a N, G N, D, G, H, S, T0.8 4  82b S S, N, D, G, R, T 1 6  82c L L, P, V 0.1 2 83 Hallmarkresidue: R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Q or T; preferably K or 0.9 7 R;most preferably K 84 Hallmark residue: P⁽⁵⁾, A, D, L, R, S, T, V;preferably P 0.7 6 85 E, G E, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S0.2 3 88 A A, G, S 0.3 2 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y,F 0 1 91 Y, H Y, D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, T A, N,G, H, K, N, R, S, T, V, Y 1.4 10 94 K, R, T A, V, C, F, G, I, K, L, R, Sor T 1.6 9

TABLE A-8 Non-limiting examples of amino acid residues in FR4 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 103 Hallmarkresidue: W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S; preferably W 0.4 2 104 Hallmark residue: Gor D; preferably G 0.1 1 105 Q, R Q, E, K, P, R 0.6 4 106 G G 0.1 1 107T T, A, I 0.3 2 108 Hallmark residue: Q, L⁽⁷⁾ or R; preferably Q or L⁽⁷⁾0.4 3 109 V V 0.1 1 110 T T, I, A 0.2 1 111 V V, A, I 0.3 2 112 S S, F0.3 1 113 S S, A, L, P, T 0.4 3

Thus, in another preferred, but not limiting aspect, a Nanobody of theinvention can be defined as an amino acid sequence with the (general)structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   -   i) one or more of the amino acid residues at positions 11, 37,        44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat        numbering are chosen from the Hallmark residues mentioned in        Table A-3;        and in which:    -   ii) CDR1, CDR2 and CDR3 are as defined herein, and are        preferably as defined according to one of the preferred aspects        herein, and are more preferably as defined according to one of        the more preferred aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In particular, a Nanobody of the invention can be an amino acid sequencewith the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   -   i) (preferably) one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3 (it being understood that V_(HH)        sequences will contain one or more Hallmark residues; and that        partially humanized Nanobodies will usually, and preferably,        [still] contain one or more Hallmark residues [although it is        also within the scope of the invention to provide - where        suitable in accordance with the invention—partially humanized        Nanobodies in which all Hallmark residues, but not one or more        of the other amino acid residues, have been humanized]; and that        in fully humanized Nanobodies, where suitable in accordance with        the invention, all amino acid residues at the positions of the        Hallmark residues will be amino acid residues that occur in a        human V_(H)3 sequence. As will be clear to the skilled person        based on the disclosure herein that such V_(HH) sequences, such        partially humanized Nanobodies with at least one Hallmark        residue, such partially humanized Nanobodies without Hallmark        residues and such fully humanized Nanobodies all form aspects of        this invention);        and in which:    -   ii) said amino acid sequence has at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 1 to 22, in which for the purposes of determining the        degree of amino acid identity, the amino acid residues that form        the CDR sequences (indicated with X in the sequences of SEQ ID        NO's: 1 to 22) are disregarded;        and in which:    -   iii) CDR1, CDR2 and CDR3 are as defined herein, and are        preferably as defined according to one of the preferred aspects        herein, and are more preferably as defined according to one of        the more preferred aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

TABLE A-9 Representative amino acid sequences for Nanobodies of theKERE, GLEW and P,R,S 103 group. KERE sequence no. 1 SEQ ID NO: 1EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence no. 2 SEQ IDNO: 2 QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTISRDTASNRGYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 3 SEQ IDNO: 3 AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTISRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence no. 4 SEQ IDNO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTISRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 5 SEQ IDNO: 5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence no. 6 SEQ IDNO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFTISSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence no. 7 SEQ IDNO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTIARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence no. 8 SEQ IDNO: 8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTISTDNAKNTVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence no. 9 SEQ IDNO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTISGDNAKRAIYLQMNNLKPDDTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence no. 10 SEQ IDNO: 10 QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTISRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 11 SEQ IDNO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTIARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence no. 12 SEQ IDNO:12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTISRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 13 SEQ IDNO:13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFTIISRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 14 SEQID NO: 14 AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTIVSRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence no. 15 SEQID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTISRDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence no. 16 SEQ IDNO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence no. 1 SEQ IDNO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTISRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence no. 2 SEQ IDNO: 18 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence no. 3 SEQID NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTISRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 sequence no. 1SEQ ID NO: 20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103 sequence no. 2SEQ ID NO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFTISRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R, S 103 sequence no. 3SEQ ID NO: 22 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS The CDR's are indicatedwith XXXX

In particular, a Nanobody of the invention of the KERE group can be anamino acid sequence with the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which:

-   -   i) the amino acid residue at position 45 according to the Kabat        numbering is a charged amino acid (as defined herein) or a        cysteine residue, and position 44 is preferably an E;        and in which:    -   ii) FR1 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-10 Representative FW1 sequences for Nanobodies of theKERE-group. KERE FW1 sequence no. 1 SEQ ID NO: 23QVQRVESGGGLVQAGGSLRLSCAASGRTSS KERE FW1 sequence no. 2 SEQ ID NO: 24QVQLVESGGGLVQTGDSLSLSCSASGRTFS KERE FW1 sequence no. 3 SEQ ID NO: 25QVKLEESGGGLVQAGDSLRLSCAATGRAFG KERE FW1 sequence no. 4 SEQ ID NO: 26AVQLVESGGGLVQPGESLGLSCVASGRDFV KERE FW1 sequence no. 5 SEQ ID NO: 27EVQLVESGGGLVQAGGSLRLSCEVLGRTAG KERE FW1 sequence no. 6 SEQ ID NO: 28QVQLVESGGGWVQPGGSLRLSCAASETILS KERE FW1 sequence no. 7 SEQ ID NO: 29QVQLVESGGGTVQPGGSLNLSCVASGNTFN KERE FW1 sequence no. 8 SEQ ID NO: 30EVQLVESGGGLAQPGGSLQLSCSAPGFTLD KERE FW1 sequence no. 9 SEQ ID NO: 31AQELEESGGGLVQAGGSLRLSCAASGRTFNand in which:

-   -   iii) FR2 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-11 Representative FW2 sequences for Nanobodies of theKERE-group. KERE FW2 SEQ ID NO: 41 WFRQAPGKEREFVA sequence no. 1 KEREFW2 SEQ ID NO: 42 WFRQTPGREREFVA sequence no. 2 KERE FW2 SEQ ID NO: 43WYRQAPGKQREMVA sequence no. 3 KERE FW2 SEQ ID NO: 44 WYRQGPGKQRELVAsequence no. 4 KERE FW2 SEQ ID NO: 45 WIRQAPGKEREGVS sequence no. 5 KEREFW2 SEQ ID NO: 46 WFREAPGKEREGIS sequence no. 6 KERE FW2 SEQ ID NO: 47WYRQAPGKERDLVA sequence no. 7 KERE FW2 SEQ ID NO: 48 WFRQAPGKQREEVSsequence no. 8 KERE FW2 SEQ ID NO: 49 WFRQPPGKVREFVG sequence no. 9and in which:

-   -   iv) FR3 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-12 Representative FW3 sequences for Nanobodies of theKERE-group. KERE FW3 SEQ ID RFTISRDNAKNTVYLQMNSLKPEDTAVYRCYF sequenceNO: 50 no. 1 KERE FW3 SEQ ID RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAA sequenceNO: 51 no. 2 KERE FW3 SEQ ID RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAA sequenceNO: 52 no. 3 KERE FW3 SEQ ID RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAA sequenceNO: 53 no. 4 KERE FW3 SEQ ID RLTISRDNAVDTMYLQMNSLKPEDTAVYYCAA sequenceNO: 54 no. 5 KERE FW3 SEQ ID RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAA sequenceNO: 55 no. 6 KERE FW3 SEQ ID RFTISKDSGKNTVYLQMTSLKPEDTAVYYCAT sequenceNO: 56 no. 7 KERE FW3 SEQ ID RFTISRDSAKNMMYLQMNNLKPQDTAVYYCAA sequenceNO: 57 no. 8 KERE FW3 SEQ ID RFTISRENDKSTVYLQLNSLKPEDTAVYYCAA sequenceNO: 58 no. 9 KERE FW3 SEQ ID RFTISRDYAGNTAYLQMNSLKPEDTGVYYCAT sequenceNO: 59 no. 10and in which:

-   -   v) FR4 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-13 Representative FW4 sequences for Nanobodies of theKERE-group. KERE FW4 sequence no. 1 SEQ ID NO: 60 WGQGTQVTVSS KERE FW4sequence no. 2 SEQ ID NO: 61 WGKGTLVTVSS KERE FW4 sequence no. 3 SEQ IDNO: 62 RGQGTRVTVSS KERE FW4 sequence no. 4 SEQ ID NO: 63 WGLGTQVTISSand in which:

-   -   vi) CDR1, CDR2 and CDR3 are as defined herein, and are        preferably as defined according to one of the preferred aspects        herein, and are more preferably as defined according to one of        the more preferred aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

Also, the above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.With regard to framework 1, it will be clear to the skilled person that,when an amino acid sequence as outlined above is generated by expressionof a nucleotide sequence, the first four amino acid sequences (i.e.amino acid residues 1-4 according to the Kabat numbering) may often bedetermined by the primer(s) that have been used to generate said nucleicacid. Thus, for determining the degree of amino acid identity, the firstfour amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27to 30 are according to the Kabat numbering considered to be part of theframework regions (and not the CDR's), it has been found by analysis ofa database of more than 1000 V_(HH) sequences that the positions 27 to30 have a variability (expressed in terms of V_(HH) entropy and V_(HH)variability—see Tables A-5 to A-8) that is much greater than thevariability on positions 1 to 26. Because of this, for determining thedegree of amino acid identity, the amino acid residues at positions 27to 30 are preferably also disregarded.

In view of this, a Nanobody of the KERE class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   -   i) the amino acid residue at position 45 according to the Kabat        numbering is a charged amino acid (as defined herein) or a        cysteine residue, and position 44 is preferably an E;        and in which:    -   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of        the Kabat numbering, has at least 80% amino acid identity with        at least one of the following amino acid sequences:

TABLE A-14 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the KERE-group. KERE FW1 se- SEQ ID NO: 32VESGGGLVQPGGSLRLSCAASG quence no. 10 KERE FW1 se- SEQ ID NO: 33VDSGGGLVQAGDSLKLSCALTG quence no. 11 KERE FW1 se- SEQ ID NO: 34VDSGGGLVQAGDSLRLSCAASG quence no. 12 KERE FW1 se- SEQ ID NO: 35VDSGGGLVEAGGSLRLSCQVSE quence no. 13 KERE FW1 se- SEQ ID NO: 36QDSGGGSVQAGGSLKLSCAASG quence no. 14 KERE FW1 se- SEQ ID NO: 37VQSGGRLVQAGDSLRLSCAASE quence no. 15 KERE FW1 se- SEQ ID NO: 38VESGGTLVQSGDSLKLSCASST quence no. 16 KERE FW1 se- SEQ ID NO: 39MESGGDSVQSGGSLTLSCVASG quence no. 17 KERE FW1 se- SEQ ID NO: 40QASGGGLVQAGGSLRLSCSASV quence no. 18and in which:

-   -   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and        FR4 of Nanobodies of the KERE-class;        and in which:    -   iv) CDR1, CDR2 and CDR3 are as defined herein, and are        preferably as defined according to one of the preferred aspects        herein, and are more preferably as defined according to one of        the more preferred aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

A Nanobody of the GLEW class may be an amino acid sequence that iscomprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   -   i) preferably, when the Nanobody of the GLEW-class is a        non-humanized Nanobody, the amino acid residue in position 108        is Q;    -   ii) FR1 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-15 Representative FW1 sequences for Nanobodies of theGLEW-group. GLEW FW1 se- SEQ ID QVQLVESGGGLVQPGGSLRLSCAASGFTFS quenceno. 1 NO: 64 GLEW FW1 se- SEQ ID EVHLVESGGGLVRPGGSLRLSCAAFGFIFK quenceno. 2 NO: 65 GLEW FW1 se- SEQ ID QVKLEESGGGLAQPGGSLRLSCVASGFTFS quenceno. 3 NO: 66 GLEW FW1 se- SEQ ID EVQLVESGGGLVQPGGSLRLSCVCVSSGCT quenceno. 4 NO: 67 GLEW FW1 se- SEQ ID EVQLVESGGGLALPGGSLTLSCVFSGSTFS quenceno. 5 NO: 68and in which:

-   -   iii) FR2 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-16 Representative FW2 sequences for Nanobodies of theGLEW-group. GLEW FW2 sequence no. SEQ ID NO: 72 WVRQAPGKVLEWVS 1 GLEWFW2 sequence no. SEQ ID NO: 73 WVRRPPGKGLEWVS 2 GLEW FW2 sequence no.SEQ ID NO: 74 WVRQAPGMGLEWVS 3 GLEW FW2 sequence no. SEQ ID NO: 75WVRQAPGKEPEWVS 4 GLEW FW2 sequence no. SEQ ID NO: 76 WVRQAPGKDQEWVS 5GLEW FW2 sequence no. SEQ ID NO: 77 WVRQAPGKAEEWVS 6 GLEW FW2 sequenceno. SEQ ID NO: 78 WVRQAPGKGLEWVA 7 GLEW FW2 sequence no. SEQ ID NO: 79WVRQAPGRATEWVS 8and in which:

-   -   iv) FR3 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-17 Representative FW3 sequences for Nanobodies of theGLEW-group. GLEW FW3 SEQ ID RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVK sequenceNO: 80 no. 1 GLEW FW3 SEQ ID RFTISRDNARNTLYLQMDSLIPEDTALYYCAR sequenceNO: 81 no. 2 GLEW FW3 SEQ ID RFTSSRDNAKSTLYLQMNDLKPEDTALYYCAR sequenceNO: 82 no. 3 GLEW FW3 SEQ ID RFIISRDNAKNTLYLQMNSLGPEDTAMYYCQR sequenceNO: 83 no. 4 GLEW FW3 SEQ ID RFTASRDNAKNTLYLQMNSLKSEDTARYYCAR sequenceNO: 84 no. 5 GLEW FW3 SEQ ID RFTISRDNAKNTLYLQMDDLQSEDTAMYYCGR sequenceNO: 85 no. 6and in which:

-   -   v) FR4 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-18 Representative FW4 sequences for Nanobodies of theGLEW-group. GLEW FW4 sequence no. 1 SEQ ID NO: 86 GSQGTQVTVSS GLEW FW4sequence no. 2 SEQ ID NO: 87 LRGGTQVTVSS GLEW FW4 sequence no. 3 SEQ IDNO: 88 RGQGTLVTVSS GLEW FW4 sequence no. 4 SEQ ID NO: 89 RSRGIQVTVSSGLEW FW4 sequence no. 5 SEQ ID NO: 90 WGKGTQVTVSS GLEW FW4 sequence no.6 SEQ ID NO: 91 WGQGTQVTVSSand in which:

-   -   vi) CDR1, CDR2 and CDR3 are as defined herein, and are        preferably as defined according to one of the preferred aspects        herein, and are more preferably as defined according to one of        the more preferred aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the GLEW class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   -   i) preferably, when the Nanobody of the GLEW-class is a        non-humanized Nanobody, the amino acid residue in position 108        is Q;        and in which:    -   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of        the Kabat numbering, has at least 80% amino acid identity with        at least one of the following amino acid sequences:

TABLE A-19 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the KERE-group. GLEW FW1 SEQ ID NO: 69VESGGGLVQPGGSLRLSCAASG sequence no. 6 GLEW FW1 SEQ ID NO: 70EESGGGLAQPGGSLRLSCVASG sequence no. 7 GLEW FW1 SEQ ID NO: 71VESGGGLALPGGSLTLSCVFSG sequence no. 8and in which:

-   -   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and        FR4 of Nanobodies of the GLEW-class;        and in which:    -   iv) CDR1, CDR2 and CDR3 are as defined herein, and are        preferably as defined according to one of the preferred aspects        herein, and are more preferably as defined according to one of        the more preferred aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein. Inthe above Nanobodies, one or more of the further Hallmark residues arepreferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

A Nanobody of the P, R, S 103 class may be an amino acid sequence thatis comprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   -   i) the amino acid residue at position 103 according to the Kabat        numbering is different from W;        and in which:    -   ii) preferably the amino acid residue at position 103 according        to the Kabat numbering is P, R or S, and more preferably R;    -   and in which:    -   iii) FR1 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-20 Representative FW1 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 SEQ ID AVQLVESGGGLVQAGGSLRLSCAASGRTFS FW1sequence NO: 92 no. 1 P, R, S 103 SEQ ID QVQLQESGGGMVQPGGSLRLSCAASGFDFGFW1 sequence NO: 93 no. 2 P, R, S 103 SEQ IDEVHLVESGGGLVRPGGSLRLSCAAFGFIFK FW1 sequence NO: 94 no. 3 P, R, S 103 SEQID QVQLAESGGGLVQPGGSLKLSCAASRTIVS FW1 sequence NO: 95 no. 4 P, R, S 103SEQ ID QEHLVESGGGLVDIGGSLRLSCAASERIFS FW1 sequence NO: 96 no. 5 P, R, S103 SEQ ID QVKLEESGGGLAQPGGSLRLSCVASGFTFS FW1 sequence NO: 97 no. 6 P,R, S 103 SEQ ID EVQLVESGGGLVQPGGSLRLSCVCVSSGCT FW1 sequence NO: 98 no. 7P, R, S 103 SEQ ID EVQLVESGGGLALPGGSLTLSCVFSGSTFS FW1 sequence NO: 99no. 8and in which

-   -   iv) FR2 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-21 Representative FW2 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW2 SEQ ID NO: 102 WFRQAPGKEREFVA sequence no. 1P, R, S 103 FW2 SEQ ID NO: 103 WVRQAPGKVLEWVS sequence no. 2 P, R, S 103FW2 SEQ ID NO: 104 WVRRPPGKGLEWVS sequence no. 3 P, R, S 103 FW2 SEQ IDNO: 105 WIRQAPGKEREGVS sequence no. 4 P, R, S 103 FW2 SEQ ID NO: 106WVRQYPGKEPEWVS sequence no. 5 P, R, S 103 FW2 SEQ ID NO: 107WFRQPPGKEHEFVA sequence no. 6 P, R, S 103 FW2 SEQ ID NO: 108WYRQAPGKRTELVA sequence no. 7 P, R, S 103 FW2 SEQ ID NO: 109WLRQAPGQGLEWVS sequence no. 8 P, R, S 103 FW2 SEQ ID NO: 110WLRQTPGKGLEWVG sequence no. 9 P, R, S 103 FW2 SEQ ID NO: 111WVRQAPGKAEEFVS sequence no. 10and in which:

-   -   v) FR3 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-22 Representative FW3 sequences for Nanobodies of the P, R, S103-group. P, R, S SEQ ID RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA 103 FW3 NO:112 sequence no. 1 P, R, S SEQ ID RFTISRDNARNTLYLQMDSLIPEDTALYYCAR 103FW3 NO: 113 sequence no. 2 P, R, S SEQ IDRFTISRDNAKNEMYLQMNNLKTEDTGVYWCGA 103 FW3 NO: 114 sequence no. 3 P, R, SSEQ ID RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAA 103 FW3 NO: 115 sequence no. 4P, R, S SEQ ID RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR 103 FW3 NO: 116 sequenceno. 5 P, R, S SEQ ID RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNL 103 FW3 NO: 117sequence no. 6 P, R, S SEQ ID RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQR 103 FW3NO: 118 sequence no. 7 P, R, S SEQ ID RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAV103 FW3 NO: 119 sequence no. 8and in which:

-   -   vi) FR4 is an amino acid sequence that has at least 80% amino        acid identity with at least one of the following amino acid        sequences:

TABLE A-23 Representative FW4 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW4 sequence SEQ ID NO: 120 RGQGTQVTVSS no. 1 P,R, S 103 FW4 sequence SEQ ID NO: 121 LRGGTQVTVSS no. 2 P, R, S 103 FW4sequence SEQ ID NO: 122 GNKGTLVTVSS no. 3 P, R, S 103 FW4 sequence SEQID NO: 123 SSPGTQVTVSS no. 4 P, R, S 103 FW4 sequence SEQ ID NO: 124SSQGTLVTVSS no. 5 P, R, S 103 FW4 sequence SEQ ID NO: 125 RSRGIQVTVSSno. 6and in which:

-   -   vii) CDR1, CDR2 and CDR3 are as defined herein, and are        preferably as defined according to one of the preferred aspects        herein, and are more preferably as defined according to one of        the more preferred aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the P,R,S 103 class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   -   i) the amino acid residue at position 103 according to the Kabat        numbering is different from W;        and in which:    -   ii) preferably the amino acid residue at position 103 according        to the Kabat numbering is P, R or S, and more preferably R;        and in which:    -   iii) FR1 is an amino acid sequence that, on positions 5 to 26 of        the Kabat numbering, has at least 80% amino acid identity with        at least one of the following amino acid sequences:

TABLE A-24 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the P, R, S 103-group. P, R,S 103 SEQ ID NO: 100VESGGGLVQAGGSLRLSCAASG FW1 sequence no. 9 P, R,S 103 SEQ ID NO: 101AESGGGLVQPGGSLKLSCAASR FW1 sequence no. 10and in which:

-   -   iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and        FR4 of Nanobodies of the P,R,S 103 class;        and in which:    -   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably        as defined according to one of the preferred aspects herein, and        are more preferably as defined according to one of the more        preferred aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody as described above, in which the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 868 to 973 and/or 1044 to 1053. This degree of amino acid identitycan for example be determined by determining the degree of amino acididentity (in a manner described herein) between said Nanobody and one ormore of the sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053, inwhich the amino acid residues that form the framework regions aredisregarded. Such Nanobodies can be as further described herein.

As already mentioned herein, another preferred but non-limiting aspectof the invention relates to a Nanobody with an amino acid sequence thatis chosen from the group consisting of SEQ ID NO's: 868 to 973 and/or1044 to 1053 or from the group consisting of from amino acid sequencesthat have more than 80%, preferably more than 90%, more preferably morethan 95%, such as 99% or more sequence identity (as defined herein) withat least one of the amino acid sequences of SEQ ID NO's: 868 to 973and/or 1044 to 1053.

Also, in the above Nanobodies:

-   -   i) any amino acid substitution (when it is not a humanizing        substitution as defined herein) is preferably, and compared to        the corresponding amino acid sequence of SEQ ID NO's: 868 to 973        and/or 1044 to 1053, a conservative amino acid substitution, (as        defined herein);        and/or:    -   ii) its amino acid sequence preferably contains either only        amino acid substitutions, or otherwise preferably no more than        5, preferably no more than 3, and more preferably only 1 or 2        amino acid deletions or insertions, compared to the        corresponding amino acid sequence of SEQ ID NO's: 868 to 973        and/or 1044 to 1053;        and/or    -   iii) the CDR's may be CDR's that are derived by means of        affinity maturation, for example starting from the CDR's of to        the corresponding amino acid sequence of SEQ ID NO's: 868 to 973        and/or 1044 to 1053.

Preferably, the CDR sequences and FR sequences in the Nanobodies of theinvention are such that the Nanobodies of the invention (andpolypeptides of the invention comprising the same):

-   -   bind to a metalloproteinase from the ADAM family with a        dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or        less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more        preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association        constant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and        preferably 10⁷ to 10¹² liter/moles or more and more preferably        10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to a metalloproteinase from the ADAM family with a        k_(on)-rate of between 10²M⁻¹ s⁻¹ to about 10⁷M⁻¹ s⁻¹,        preferably between 10³M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, more preferably        between 10⁴M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, such as between 10⁵M⁻¹ s⁻¹        and 10⁷M⁻¹ s⁻¹;        and/or such that they:    -   bind to a metalloproteinase from the ADAM family with a k_(off)        rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a        near irreversible complex with a t_(1/2) of multiple days),        preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably        between 10⁻³ s⁻¹ and 10⁻⁴s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶        s⁻¹.

Preferably, CDR sequences and FR sequences present in the Nanobodies ofthe invention are such that the Nanobodies of the invention will bind toa metalloproteinase from the ADAM family with an affinity less than 500nM, preferably less than 200 nM, more preferably less than 10 nM, suchas less than 500 pM.

According to one non-limiting aspect of the invention, a Nanobody may beas defined herein, but with the proviso that it has at least “one aminoacid difference” (as defined herein) in at least one of the frameworkregions compared to the corresponding framework region of a naturallyoccurring human V_(H) domain, and in particular compared to thecorresponding framework region of DP-47. More specifically, according toone non-limiting aspect of the invention, a Nanobody may be as definedherein, but with the proviso that it has at least “one amino aciddifference” (as defined herein) at at least one of the Hallmark residues(including those at positions 108, 103 and/or 45) compared to thecorresponding framework region of a naturally occurring human V_(H)domain, and in particular compared to the corresponding framework regionof DP-47. Usually, a Nanobody will have at least one such amino aciddifference with a naturally occurring V_(H) domain in at least one ofFR2 and/or FR4, and in particular at at least one of the Hallmarkresidues in FR2 and/or FR4 (again, including those at positions 108, 103and/or 45).

Also, a humanized Nanobody of the invention may be as defined herein,but with the proviso that it has at least “one amino acid difference”(as defined herein) in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring V_(HH)domain. More specifically, according to one non-limiting aspect of theinvention, a humanized Nanobody may be as defined herein, but with theproviso that it has at least “one amino acid difference” (as definedherein) at at least one of the Hallmark residues (including those atpositions 108, 103 and/or 45) compared to the corresponding frameworkregion of a naturally occurring V_(HH) domain. Usually, a humanizedNanobody will have at least one such amino acid difference with anaturally occurring V_(HH) domain in at least one of FR2 and/or FR4, andin particular at at least one of the Hallmark residues in FR2 and/or FR4(again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scopeof the invention to use natural or synthetic analogs, mutants, variants,alleles, homologs and orthologs (herein collectively referred to as“analogs”) of the Nanobodies of the invention as defined herein, and inparticular analogs of the Nanobodies of SEQ ID NO's 868 to 973 and/or1044 to 1053. Thus, according to one aspect of the invention, the term“Nanobody of the invention” in its broadest sense also covers suchanalogs.

Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the Nanobodies of theinvention as defined herein. Such substitutions, insertions or deletionsmay be made in one or more of the framework regions and/or in one ormore of the CDR's. When such substitutions, insertions or deletions aremade in one or more of the framework regions, they may be made at one ormore of the Hallmark residues and/or at one or more of the otherpositions in the framework residues, although substitutions, insertionsor deletions at the Hallmark residues are generally less preferred(unless these are suitable humanizing substitutions as describedherein).

By means of non-limiting examples, a substitution may for example be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(HH) domain (see Tables A-5 toA-8 for some non-limiting examples of such substitutions), although theinvention is generally not limited thereto. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the Nanobody of the invention or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the Nanobody of theinvention (i.e. to the extent that the Nanobody is no longer suited forits intended use) are included within the scope of the invention. Askilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinations ofthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may for example involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

For example, and depending on the host organism used to express theNanobody or polypeptide of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art. Alternatively, substitutions or insertions may be designedso as to introduce one or more sites for attachment of functional groups(as described herein), for example to allow site-specific pegylation(again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables A-5 to A-8 above, some amino acid residuesin the framework regions are more conserved than others. Generally,although the invention in its broadest sense is not limited thereto, anysubstitutions, deletions or insertions are preferably made at positionsthat are less conserved. Also, generally, amino acid substitutions arepreferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to ametalloproteinase from the ADAM family with an affinity (suitablymeasured and/or expressed as a K_(D) value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein) that isas defined herein for the Nanobodies of the invention.

The analogs are preferably also such that they retain the favourableproperties the Nanobodies, as described herein.

Also, according to one preferred aspect, the analogs have a degree ofsequence identity of at least 70%, preferably at least 80%, morepreferably at least 90%, such as at least 95% or 99% or more; and/orpreferably have at most 20, preferably at most 10, even more preferablyat most 5, such as 4, 3, 2 or only 1 amino acid difference (as definedherein), with one of the Nanobodies of SEQ ID NOs: 868 to 973 and/or1044 to 1053.

Also, the framework sequences and CDR's of the analogs are preferablysuch that they are in accordance with the preferred aspects definedherein. More generally, as described herein, the analogs will have (a) aQ at position 108; and/or (b) a charged amino acid or a cysteine residueat position 45 and preferably an E at position 44, and more preferably Eat position 44 and R at position 45; and/or (c) P, R or S at position103.

One preferred class of analogs of the Nanobodies of the inventioncomprise Nanobodies that have been humanized (i.e. compared to thesequence of a naturally occurring Nanobody of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, for example from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparison between the sequence of a Nanobody and the sequence of anaturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resultinghumanized Nanobodies still retain the favourable properties ofNanobodies as defined herein, and more preferably such that they are asdescribed for analogs in the preceding paragraphs. A skilled person willgenerally be able to determine and select suitable humanizingsubstitutions or suitable combinations of humanizing substitutions,based on the disclosure herein and optionally after a limited degree ofroutine experimentation, which may for example involve introducing alimited number of possible humanizing substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the inventionmay become more “human-like”, while still retaining the favorableproperties of the Nanobodies of the invention as described herein. As aresult, such humanized Nanobodies may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(HH) domains. Again, based on the disclosure herein andoptionally after a limited degree of routine experimentation, theskilled person will be able to select humanizing substitutions orsuitable combinations of humanizing substitutions which optimize orachieve a desired or suitable balance between the favourable propertiesprovided by the humanizing substitutions on the one hand and thefavourable properties of naturally occurring V_(HH) domains on the otherhand.

The Nanobodies of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues (asdefined herein) or at one or more other framework residues (i.e.non-Hallmark residues) or any suitable combination thereof. Onepreferred humanizing substitution for Nanobodies of the “P,R,S-103group” or the “KERE group” is Q108 into L108. Nanobodies of the “GLEWclass” may also be humanized by a Q108 into L108 substitution, providedat least one of the other Hallmark residues contains a camelid(camelizing) substitution (as defined herein). For example, as mentionedabove, one particularly preferred class of humanized Nanobodies has GLEWor a GLEW-like sequence at positions 44-47; P, R or S (and in particularR) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se. For example, theanalogs can be obtained by providing a nucleic acid that encodes anaturally occurring V_(HH) domain, changing the codons for the one ormore amino acid residues that are to be substituted into the codons forthe corresponding desired amino acid residues (e.g. by site-directedmutagenesis or by PCR using suitable mismatch primers), expressing thenucleic acid/nucleotide sequence thus obtained in a suitable host orexpression system; and optionally isolating and/or purifying the analogthus obtained to provide said analog in essentially isolated form (e.g.as further described herein). This can generally be performed usingmethods and techniques known per se, which will be clear to the skilledperson, for example from the handbooks and references cited herein, thebackground art cited herein and/or from the further description herein.Alternatively, a nucleic acid encoding the desired analog can besynthesized in a manner known per se (for example using an automatedapparatus for synthesizing nucleic acid sequences with a predefinedamino acid sequence) and can then be expressed as described herein. Yetanother technique may involve combining one or more naturally occurringand/or synthetic nucleic acid sequences each encoding a part of thedesired analog, and then expressing the combined nucleic acid sequenceas described herein. Also, the analogs can be provided using chemicalsynthesis of the pertinent amino acid sequence using techniques forpeptide synthesis known per se, such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that theNanobodies of the invention (including their analogs) can be designedand/or prepared starting from human V_(H) sequences (i.e. amino acidsequences or the corresponding nucleotide sequences), such as forexample from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e.by introducing one or more camelizing substitutions (i.e. changing oneor more amino acid residues in the amino acid sequence of said humanV_(H) domain into the amino acid residues that occur at thecorresponding position in a V_(HH) domain), so as to provide thesequence of a Nanobody of the invention and/or so as to confer thefavourable properties of a Nanobody to the sequence thus obtained.Again, this can generally be performed using the various methods andtechniques referred to in the previous paragraph, using an amino acidsequence and/or nucleotide sequence for a human V_(H) domain as astarting point.

Some preferred, but non-limiting camelizing substitutions can be derivedfrom Tables A-5-A-8. It will also be clear that camelizing substitutionsat one or more of the Hallmark residues will generally have a greaterinfluence on the desired properties than substitutions at one or more ofthe other amino acid positions, although both and any suitablecombination thereof are included within the scope of the invention. Forexample, it is possible to introduce one or more camelizingsubstitutions that already confer at least some the desired properties,and then to introduce further camelizing substitutions that eitherfurther improve said properties and/or confer additional favourableproperties. Again, the skilled person will generally be able todetermine and select suitable camelizing substitutions or suitablecombinations of camelizing substitutions, based on the disclosure hereinand optionally after a limited degree of routine experimentation, whichmay for example involve introducing a limited number of possiblecamelizing substitutions and determining whether the favourableproperties of Nanobodies are obtained or improved (i.e. compared to theoriginal V_(H) domain). Generally, however, such camelizingsubstitutions are preferably such that the resulting an amino acidsequence at least contains (a) a Q at position 108; and/or (b) a chargedamino acid or a cysteine residue at position 45 and preferably also an Eat position 44, and more preferably E at position 44 and R at position45; and/or (c) P, R or S at position 103; and optionally one or morefurther camelizing substitutions. More preferably, the camelizingsubstitutions are such that they result in a Nanobody of the inventionand/or in an analog thereof (as defined herein), such as in a humanizedanalog and/or preferably in an analog that is as defined in thepreceding paragraphs.

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the Nanobodies of the invention asdefined herein, and in particular parts or fragments of the Nanobodiesof SEQ ID NO's: 868 to 973 and/or 1044 to 1053. Thus, according to oneaspect of the invention, the term “Nanobody of the invention” in itsbroadest sense also covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies of the invention(including analogs thereof) have amino acid sequences in which, comparedto the amino acid sequence of the corresponding full length Nanobody ofthe invention (or analog thereof), one or more of the amino acidresidues at the N-terminal end, one or more amino acid residues at theC-terminal end, one or more contiguous internal amino acid residues, orany combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to ametalloproteinase from the ADAM family with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein) that isas defined herein for the Nanobodies of the invention.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theamino acid sequence of the corresponding full length Nanobody of theinvention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect,such a part or fragment comprises at least CDR3, such as FR3, CDR3 andFR4 of the corresponding full length Nanobody of the invention, i.e. asfor example described in the International application WO 03/050531(Lasters et al.).

As already mentioned above, it is also possible to combine two or moreof such parts or fragments (i.e. from the same or different Nanobodiesof the invention), i.e. to provide an analog (as defined herein) and/orto provide further parts or fragments (as defined herein) of a Nanobodyof the invention. It is for example also possible to combine one or moreparts or fragments of a Nanobody of the invention with one or more partsor fragments of a human V_(H) domain.

According to one preferred aspect, the parts or fragments have a degreeof sequence identity of at least 50%, preferably at least 60%, morepreferably at least 70%, even more preferably at least 80%, such as atleast 90%, 95% or 99% or more with one of the Nanobodies of SEQ ID NOs868 to 973 and/or 1044 to 1053.

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized Nanobody of theinvention, and then expressing the nucleic acid thus obtained in amanner known per se (e.g. as described herein). Alternatively, nucleicacids encoding such parts or fragments can be obtained by suitablyrestricting a nucleic acid that encodes a full-sized Nanobody of theinvention or by synthesizing such a nucleic acid in a manner known perse. Parts or fragments may also be provided using techniques for peptidesynthesis known per se.

The invention in its broadest sense also comprises derivatives of theNanobodies of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.g.enzymatical) modification, of the Nanobodies of the invention and/or ofone or more of the amino acid residues that form the Nanobodies of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the Nanobody sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the Nanobody of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that increase the half-life, the solubility and/or theabsorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to,fluorescent labels (such as fluorescein, isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, andfluorescamine and fluorescent metals such as ¹⁵²Eu or others metals fromthe lanthanide series), phosphorescent labels, chemiluminescent labelsor bioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metal chelates ormetallic cations (for example metallic cations such as ^(99m)Tc, ¹²³I,¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metalliccations that are particularly suited for use in in vivo, in vitro or insitu diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe), as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels will beclear to the skilled person, and for example include moieties that canbe detected using NMR or ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, EIA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethylenetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to ametalloproteinase from the ADAM family with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein) that isas defined herein for the Nanobodies of the invention.

As mentioned above, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least oneNanobody of the invention. By “essentially consist of” is meant that theamino acid sequence of the polypeptide of the invention either isexactly the same as the amino acid sequence of a Nanobody of theinvention or corresponds to the amino acid sequence of a Nanobody of theinvention which has a limited number of amino acid residues, such as1-20 amino acid residues, for example 1-10 amino acid residues andpreferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 aminoacid residues, added at the amino terminal end, at the carboxy terminalend, or at both the amino terminal end and the carboxy terminal end ofthe amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, such amino acidresidues:

-   -   can comprise an N-terminal Met residue, for example as result of        expression in a heterologous host cell or host organism.    -   may form a signal sequence or leader sequence that directs        secretion of the Nanobody from a host cell upon synthesis.        Suitable secretory leader peptides will be clear to the skilled        person, and may be as further described herein. Usually, such a        leader sequence will be linked to the N-terminus of the        Nanobody, although the invention in its broadest sense is not        limited thereto;    -   may form a sequence or signal that allows the Nanobody to be        directed towards and/or to penetrate or enter into specific        organs, tissues, cells, or parts or compartments of cells,        and/or that allows the Nanobody to penetrate or cross a        biological barrier such as a cell membrane, a cell layer such as        a layer of epithelial cells, a tumor including solid tumors, or        the blood-brain-barrier. Examples of such amino acid sequences        will be clear to the skilled person. Some non-limiting examples        are the small peptide vectors (“Pep-trans vectors”) described in        WO 03/026700 and in Temsamani et al., Expert Opin. Biol. Ther.,        1, 773 (2001); Temsamani and Vidal, Drug Discov. Today, 9,        1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131        (2001), and the membrane translocator sequence described by Zhao        et al., Apoptosis, 8, 631-637 (2003). C-terminal and N-terminal        amino acid sequences for intracellular targeting of antibody        fragments are for example described by Cardinale et al.,        Methods, 34, 171 (2004). Other suitable techniques for        intracellular targeting involve the expression and/or use of        so-called “intrabodies” comprising a Nanobody of the invention,        as mentioned below;    -   may form a “tag”, for example an amino acid sequence or residue        that allows or facilitates the purification of the Nanobody, for        example using affinity techniques directed against said sequence        or residue. Thereafter, said sequence or residue may be removed        (e.g. by chemical or enzymatical cleavage) to provide the        Nanobody sequence (for this purpose, the tag may optionally be        linked to the Nanobody sequence via a cleavable linker sequence        or contain a cleavable motif). Some preferred, but non-limiting        examples of such residues are multiple histidine residues,        glutathione residues and a myc-tag (see for example SEQ ID NO:31        of WO 06/12282).    -   may be one or more amino acid residues that have been        functionalized and/or that can serve as a site for attachment of        functional groups. Suitable amino acid residues and functional        groups will be clear to the skilled person and include, but are        not limited to, the amino acid residues and functional groups        mentioned herein for the derivatives of the Nanobodies of the        invention.

According to another aspect, a polypeptide of the invention comprises aNanobody of the invention, which is fused at its amino terminal end, atits carboxy terminal end, or both at its amino terminal end and at itscarboxy terminal end to at least one further amino acid sequence, i.e.so as to provide a fusion protein comprising said Nanobody of theinvention and the one or more further amino acid sequences. Such afusion will also be referred to herein as a “Nanobody fusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further amino acid sequences may ormay not change, alter or otherwise influence the (biological) propertiesof the Nanobody, and may or may not add further functionality to theNanobody or the polypeptide of the invention. Preferably, the furtheramino acid sequence is such that it confers one or more desiredproperties or functionalities to the Nanobody or the polypeptide of theinvention.

For example, the further amino acid sequence may also provide a secondbinding site, which binding site may be directed against any desiredprotein, polypeptide, antigen, antigenic determinant or epitope(including but not limited to the same protein, polypeptide, antigen,antigenic determinant or epitope against which the Nanobody of theinvention is directed, or a different protein, polypeptide, antigen,antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005),

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the polypeptides of the invention,compared to the Nanobody of the invention per se. Some non-limitingexamples of such amino acid sequences are serum proteins, such as humanserum albumin (see for example WO 00/27435) or haptenic molecules (forexample haptens that are recognized by circulating antibodies, see forexample WO 98/22141).

In particular, it has been described in the art that linking fragmentsof immunoglobulins (such as V_(H) domains) to serum albumin or tofragments thereof can be used to increase the half-life. Reference isfor made to WO 00/27435 and WO 01/077137). According to the invention,the Nanobody of the invention is preferably either directly linked toserum albumin (or to a suitable fragment thereof) or via a suitablelinker, and in particular via a suitable peptide linked so that thepolypeptide of the invention can be expressed as a genetic fusion(protein). According to one specific aspect, the Nanobody of theinvention may be linked to a fragment of serum albumin that at leastcomprises the domain III of serum albumin or part thereof. Reference isfor example made to the U.S. provisional application 60/788,256 ofAblynx N.V. entitled “Albumin derived amino acid sequence, use thereoffor increasing the half-life of therapeutic proteins and of othertherapeutic proteins and entities, and constructs comprising the same”filed on Mar. 31, 2006 (see also PCT/EP2007/002817).

Alternatively, the further amino acid sequence may provide a secondbinding site or binding unit that is directed against a serum protein(such as, for example, human serum albumin or another serum protein suchas IgG), so as to provide increased half-life in serum. Such amino acidsequences for example include the Nanobodies described below, as well asthe small peptides and binding proteins described in WO 91/01743, WO01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41);4926-42, 2005, as well as to EP 0 368 684, as well as to the followingthe U.S. provisional application 60/843,349 (see alsoPCT/EP2007/059475), 60/850,774 (see also PCT/EP2007/060849), 60/850,775(see also PCT/EP2007/060850) by Ablynx N.V. mentioned herein USprovisional application of Ablynx N.V. entitled “Peptides capable ofbinding to serum proteins” filed on Dec. 5, 2006 (see alsoPCT/EP2007/063348).

Such amino acid sequences may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such amino acidsequences may be amino acid sequences that are directed against (human)serum albumin and amino acid sequences that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see for example WO 06/0122787) and/or amino acidsequences that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see againfor example WO 06/0122787); amino acid sequences that have or canprovide an increased half-life (see for example the U.S. provisionalapplication 60/843,349 by Ablynx N.V. entitled “Serum albumin bindingproteins with long half-lives” filed on Sep. 8, 2006; see alsoPCT/EP2007/059475); amino acid sequences against human serum albuminthat are cross-reactive with serum albumin from at least one species ofmammal, and in particular with at least one species of primate (such as,without limitation, monkeys from the genus Macaca (such as, and inparticular, cynomolgus monkeys (Macaca fascicularis) and/or rhesusmonkeys (Macaca mulatta)) and baboon (Papio ursinus), reference is againmade to the U.S. provisional application 60/843,349 andPCT/EP2007/059475); amino acid sequences that can bind to serum albuminin a pH independent manner (see for example the U.S. provisionalapplication 60/850,774 by Ablynx N.V. entitled “Amino acid sequencesthat bind to serum proteins in a manner that is essentially independentof the pH, compounds comprising the same, and uses thereof”, filed onOct. 11, 2006; see also and PCT/EP2007/059475) and/or amino acidsequences that are conditional binders (see for example the U.S.provisional application 60/850,775 by Ablynx N.V. entitled “Amino acidsequences that bind to a desired molecule in a conditional manner”,filed on Oct. 11, 2006; see also PCT/EP2007/060850).

According to another aspect, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a Nanobody of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain or to a natural or syntheticanalog of a V_(H) or V_(L) domain, again optionally via a linkersequence (including but not limited to other (single) domain antibodies,such as the dAb's described by Ward et al.).

The at least one Nanobody may also be linked to one or more (preferablyhuman) C_(H)1, C_(H)2 and/or C_(H)3 domains, optionally via a linkersequence. For instance, a Nanobody linked to a suitable C_(H)1 domaincould for example be used—together with suitable light chains—togenerate antibody fragments/structures analogous to conventional Fabfragments or F(ab′)₂ fragments, but in which one or (in case of anF(ab′)₂ fragment) one or both of the conventional V_(H) domains havebeen replaced by a Nanobody of the invention. Also, two Nanobodies couldbe linked to a C_(H)3 domain (optionally via a linker) to provide aconstruct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, oneor more Nanobodies of the invention may be linked (optionally via asuitable linker or hinge region) to one or more constant domains (forexample, 2 or 3 constant domains that can be used as part of/to form anFc portion), to an Fc portion and/or to one or more antibody parts,fragments or domains that confer one or more effector functions to thepolypeptide of the invention and/or may confer the ability to bind toone or more Fc receptors. For example, for this purpose, and withoutbeing limited thereto, the one or more further amino acid sequences maycomprise one or more C_(H)2 and/or C_(H)3 domains of an antibody, suchas from a heavy chain antibody (as described herein) and more preferablyfrom a conventional human 4-chain antibody; and/or may form (part of)and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3 orIgG4), from IgE or from another human Ig such as IgA, IgD or IgM. Forexample, WO 94/04678 describes heavy chain antibodies comprising aCamelid V_(HH) domain or a humanized derivative thereof (i.e. aNanobody), in which the Camelidae C_(H)2 and/or C_(H)3 domain have beenreplaced by human C_(H)2 and C_(H)3 domains, so as to provide animmunoglobulin that consists of 2 heavy chains each comprising aNanobody and human C_(H)2 and C_(H)3 domains (but no C_(H)1 domain),which immunoglobulin has the effector function provided by the C_(H)2and C_(H)3 domains and which immunoglobulin can function without thepresence of any light chains. Other amino acid sequences that can besuitably linked to the Nanobodies of the invention so as to provide aneffector function will be clear to the skilled person, and may be chosenon the basis of the desired effector function(s). Reference is forexample made to WO 04/058820, WO 99/42077, WO 02/056910 and WO05/017148, as well as the review by Holliger and Hudson, supra; and tothe non-prepublished US provisional application by Ablynx N.V. entitled“Constructs comprising single variable domains and an Fc portion derivedfrom IgE” which has a filing date of Dec. 4, 2007. Coupling of aNanobody of the invention to an Fc portion may also lead to an increasedhalf-life, compared to the corresponding Nanobody of the invention. Forsome applications, the use of an Fc portion and/or of constant domains(i.e. C_(H)2 and/or C_(H)3 domains) that confer increased half-lifewithout any biologically significant effector function may also besuitable or even preferred. Other suitable constructs comprising one ormore Nanobodies and one or more constant domains with increasedhalf-life in vivo will be clear to the skilled person, and may forexample comprise two Nanobodies linked to a C_(H)3 domain, optionallyvia a linker sequence. Generally, any fusion protein or derivatives withincreased half-life will preferably have a molecular weight of more than50 kD, the cut-off value for renal absorption.

In another one specific, but non-limiting, aspect, in order to form apolypeptide of the invention, one or more amino acid sequences of theinvention may be linked (optionally via a suitable linker or hingeregion) to naturally occurring, synthetic or semisynthetic constantdomains (or analogs, variants, mutants, parts or fragments thereof) thathave a reduced (or essentially no) tendency to self-associate intodimers (i.e. compared to constant domains that naturally occur inconventional 4-chain antibodies). Such monomeric (i.e. notself-associating) Fc chain variants, or fragments thereof, will be clearto the skilled person. For example, Helm et al., J Biol Chem 1996 2717494, describe monomeric Fcε chain variants that can be used in thepolypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they arestill capable of binding to the complement or the relevant Fcreceptor(s) (depending on the Fc portion from which they are derived),and/or such that they still have some or all of the effector functionsof the Fc portion from which they are derived (or at a reduced levelstill suitable for the intended use). Alternatively, in such apolypeptide chain of the invention, the monomeric Fc chain may be usedto confer increased half-life upon the polypeptide chain, in which casethe monomeric Fc chain may also have no or essentially no effectorfunctions.

Bivalent/multivalent, bispecific/multispecific orbiparatopic/multiparatopic polypeptides of the invention may also belinked to Fc portions, in order to provide polypeptide constructs of thetype that is described in the non-prepublished US provisionalapplication entitled “immunoglobulin constructs” filed on Dec. 4, 2007.

The further amino acid sequences may also form a signal sequence orleader sequence that directs secretion of the Nanobody or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro-form of the polypeptide of theinvention, depending on the host cell used to express the polypeptide ofthe invention).

The further amino acid sequence may also form a sequence or signal thatallows the Nanobody or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows theNanobody or polypeptide of the invention to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Suitable examples of such amino acid sequences willbe clear to the skilled person, and for example include, but are notlimited to, the “Peptrans” vectors mentioned above, the sequencesdescribed by Cardinale et al. and the amino acid sequences and antibodyfragments known per se that can be used to express or produce theNanobodies and polypeptides of the invention as so-called “intrabodies”,for example as described in WO 94/02610, WO 95/22618, U.S. Pat. No.7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and inCattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Developmentand Applications. Landes and Springer-Verlag; and in Kontermann, Methods34, (2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation of such acell, the Nanobodies of the invention may also be linked to a(cyto)toxic protein or polypeptide. Examples of such toxic proteins andpolypeptides which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic polypeptide of the invention will beclear to the skilled person and can for example be found in the priorart cited above and/or in the further description herein. One example isthe so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or morefurther amino acid sequences comprise at least one further Nanobody, soas to provide a polypeptide of the invention that comprises at leasttwo, such as three, four, five or more Nanobodies, in which saidNanobodies may optionally be linked via one or more linker sequences (asdefined herein). Polypeptides of the invention that comprise two or moreNanobodies, of which at least one is a Nanobody of the invention, willalso be referred to herein as “multivalent” polypeptides of theinvention, and the Nanobodies present in such polypeptides will also bereferred to herein as being in a “multivalent format”. For example a“bivalent” polypeptide of the invention comprises two Nanobodies,optionally linked via a linker sequence, whereas a “trivalent”polypeptide of the invention comprises three Nanobodies, optionallylinked via two linker sequences; etc.; in which at least one of theNanobodies present in the polypeptide, and up to all of the Nanobodiespresent in the polypeptide, is/are a Nanobody of the invention.

In a multivalent polypeptide of the invention, the two or moreNanobodies may be the same or different, and may be directed against thesame antigen or antigenic determinant (for example against the samepart(s) or epitope(s) or against different parts or epitopes) or mayalternatively be directed against different antigens or antigenicdeterminants; or any suitable combination thereof. For example, abivalent polypeptide of the invention may comprise (a) two identicalNanobodies; (b) a first Nanobody directed against a first antigenicdeterminant of a protein or antigen and a second Nanobody directedagainst the same antigenic determinant of said protein or antigen whichis different from the first Nanobody; (c) a first Nanobody directedagainst a first antigenic determinant of a protein or antigen and asecond Nanobody directed against another antigenic determinant of saidprotein or antigen; or (d) a first Nanobody directed against a firstprotein or antigen and a second Nanobody directed against a secondprotein or antigen (i.e. different from said first antigen). Similarly,a trivalent polypeptide of the invention may, for example and withoutbeing limited thereto. comprise (a) three identical Nanobodies; (b) twoidentical Nanobody against a first antigenic determinant of an antigenand a third Nanobody directed against a different antigenic determinantof the same antigen; (c) two identical Nanobody against a firstantigenic determinant of an antigen and a third Nanobody directedagainst a second antigen different from said first antigen; (d) a firstNanobody directed against a first antigenic determinant of a firstantigen, a second Nanobody directed against a second antigenicdeterminant of said first antigen and a third Nanobody directed againsta second antigen different from said first antigen; or (e) a firstNanobody directed against a first antigen, a second Nanobody directedagainst a second antigen different from said first antigen, and a thirdNanobody directed against a third antigen different from said first andsecond antigen.

Polypeptides of the invention that contain at least two Nanobodies, inwhich at least one Nanobody is directed against a first antigen (i.e.against a metalloproteinase from the ADAM family,) and at least oneNanobody is directed against a second antigen (i.e. different from ametalloproteinase from the ADAM family,), will also be referred to as“multispecific” polypeptides of the invention, and the Nanobodiespresent in such polypeptides will also be referred to herein as being ina “multispecific format”. Thus, for example, a “bispecific” polypeptideof the invention is a polypeptide that comprises at least one Nanobodydirected against a first antigen (i.e. a metalloproteinase from the ADAMfamily,) and at least one further Nanobody directed against a secondantigen (i.e. different from a metalloproteinase from the ADAM family,),whereas a “trispecific” polypeptide of the invention is a polypeptidethat comprises at least one Nanobody directed against a first antigen(i.e. a metalloproteinase from the ADAM family,), at least one furtherNanobody directed against a second antigen (i.e. different from ametalloproteinase from the ADAM family,) and at least one furtherNanobody directed against a third antigen (i.e. different from both ametalloproteinase from the ADAM family, and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against ametalloproteinase from the ADAM family, and a second Nanobody directedagainst a second antigen, in which said first and second Nanobody mayoptionally be linked via a linker sequence (as defined herein); whereasa trispecific polypeptide of the invention in its simplest form is atrivalent polypeptide of the invention (as defined herein), comprising afirst Nanobody directed against a metalloproteinase from the ADAMfamily, a second Nanobody directed against a second antigen and a thirdNanobody directed against a third antigen, in which said first, secondand third Nanobody may optionally be linked via one or more, and inparticular one and more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise at least one Nanobody againsta metalloproteinase from the ADAM family, and any number of Nanobodiesdirected against one or more antigens different from a metalloproteinasefrom the ADAM family.

Furthermore, although it is encompassed within the scope of theinvention that the specific order or arrangement of the variousNanobodies in the polypeptides of the invention may have some influenceon the properties of the final polypeptide of the invention (includingbut not limited to the affinity, specificity or avidity for ametalloproteinase from the ADAM family, or against the one or more otherantigens), said order or arrangement is usually not critical and may besuitably chosen by the skilled person, optionally after some limitedroutine experiments based on the disclosure herein. Thus, when referenceis made to a specific multivalent or multispecific polypeptide of theinvention, it should be noted that this encompasses any order orarrangements of the relevant Nanobodies, unless explicitly indicatedotherwise.

Finally, it is also within the scope of the invention that thepolypeptides of the invention contain two or more Nanobodies and one ormore further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans,Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to forexample WO 96/34103 and WO 99/23221. Some other examples of somespecific multispecific and/or multivalent polypeptide of the inventioncan be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptideof the invention comprises at least one Nanobody of the invention and atleast one Nanobody that provides for an increased half-life. SuchNanobodies may for example be Nanobodies that are directed against aserum protein, and in particular a human serum protein, such as humanserum albumin, thyroxine-binding protein, (human) transferrin,fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one ofthe serum proteins listed in WO 04/003019. Of these, Nanobodies that canbind to serum albumin (and in particular human serum albumin) or to IgG(and in particular human IgG, see for example Nanobody VH-1 described inthe review by Muyldermans, supra) are particularly preferred (althoughfor example, for experiments in mice or primates, Nanobodies against orcross-reactive with mouse serum albumin (MSA) or serum albumin from saidprimate, respectively, can be used. However, for pharmaceutical use,Nanobodies against human serum albumin or human IgG will usually bepreferred). Nanobodies that provide for increased half-life and that canbe used in the polypeptides of the invention include the Nanobodiesdirected against serum albumin that are described in WO 04/041865, in WO06/122787 and in the further patent applications by Ablynx N.V., such asthose mentioned above.

For example, the some preferred Nanobodies that provide for increasedhalf-life for use in the present invention include Nanobodies that canbind to amino acid residues on (human) serum albumin that are notinvolved in binding of serum albumin to FcRn (see for example WO06/0122787); Nanobodies that are capable of binding to amino acidresidues on serum albumin that do not form part of domain III of serumalbumin (see for example WO 06/0122787); Nanobodies that have or canprovide an increased half-life (see for example the U.S. provisionalapplication 60/843,349 by Ablynx N.V mentioned herein; see alsoPCT/EP2007/059475); Nanobodies against human serum albumin that arecross-reactive with serum albumin from at least one species of mammal,and in particular with at least one species of primate (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus)) (see for example the U.S.provisional application 60/843,349 by Ablynx N.V. see alsoPCT/EP2007/059475); Nanobodies that can bind to serum albumin in a pHindependent manner (see for example the U.S. provisional application60/850,774 by Ablynx N.V. mentioned herein) and/or Nanobodies that areconditional binders (see for example the U.S. provisional application60/850,775 by Ablynx N.V.; see also PCT/EP2007/060850).

Some particularly preferred Nanobodies that provide for increasedhalf-life and that can be used in the polypeptides of the inventioninclude the Nanobodies ALB-1 to ALB-10 disclosed in WO 06/122787 (seeTables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) isparticularly preferred.

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or moreNanobodies of the invention, at least one Nanobody against human serumalbumin.

Generally, any polypeptides of the invention with increased half-lifethat contain one or more Nanobodies of the invention, and anyderivatives of Nanobodies of the invention or of such polypeptides thathave an increased half-life, preferably have a half-life that is atleast 1.5 times, preferably at least 2 times, such as at least 5 times,for example at least 10 times or more than 20 times, greater than thehalf-life of the corresponding Nanobody of the invention per se. Forexample, such a derivative or polypeptides with increased half-life mayhave a half-life that is increased with more than 1 hours, preferablymore than 2 hours, more preferably more than 6 hours, such as more than12 hours, or even more than 24, 48 or 72 hours, compared to thecorresponding Nanobody of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchderivatives or polypeptides may exhibit a serum half-life in human of atleast about 12 hours, preferably at least 24 hours, more preferably atleast 48 hours, even more preferably at least 72 hours or more. Forexample, such derivatives or polypeptides may have a half-life of atleast 5 days (such as about 5 to 10 days), preferably at least 9 days(such as about 9 to 14 days), more preferably at least about 10 days(such as about 10 to 15 days), or at least about 11 days (such as about11 to 16 days), more preferably at least about 12 days (such as about 12to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable ofbinding to one or more molecules which can increase the half-life of thepolypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and theirhalf-life increased by binding to molecules which resist degradationand/or clearance or sequestration. Typically, such molecules arenaturally occurring proteins which themselves have a long half-life invivo.

Another preferred, but non-limiting example of a multispecificpolypeptide of the invention comprises at least one Nanobody of theinvention and at least one Nanobody that directs the polypeptide of theinvention towards, and/or that allows the polypeptide of the inventionto penetrate or to enter into specific organs, tissues, cells, or partsor compartments of cells, and/or that allows the Nanobody to penetrateor cross a biological barrier such as a cell membrane, a cell layer suchas a layer of epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Examples of such Nanobodies include Nanobodies thatare directed towards specific cell-surface proteins, markers or epitopesof the desired organ, tissue or cell (for example cell-surface markersassociated with tumor cells), and the single-domain brain targetingantibody fragments described in WO 02/057445 and WO 06/040153, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In the polypeptides of the invention, the one or more Nanobodies and theone or more polypeptides may be directly linked to each other (as forexample described in WO 99/23221) and/or may be linked to each other viaone or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each Nanobody by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))_(z), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30,GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see for example WO 06/040153 and WO 06/122825), aswell as hinge-like regions, such as the hinge regions of naturallyoccurring heavy chain antibodies or similar sequences (such as describedin WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) andGS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, poly(ethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for a metalloproteinase from the ADAM family, orfor one or more of the other antigens. Based on the disclosure herein,the skilled person will be able to determine the optimal linker(s) foruse in a specific polypeptide of the invention, optionally after somelimited routine experiments.

For example, in multivalent polypeptides of the invention that compriseNanobodies directed against a multimeric antigen (such as a multimericreceptor or other protein), the length and flexibility of the linker arepreferably such that it allows each Nanobody of the invention present inthe polypeptide to bind to the antigenic determinant on each of thesubunits of the multimer. Similarly, in a multispecific polypeptide ofthe invention that comprises Nanobodies directed against two or moredifferent antigenic determinants on the same antigen (for exampleagainst different epitopes of an antigen and/or against differentsubunits of a multimeric receptor, channel or protein), the length andflexibility of the linker are preferably such that it allows eachNanobody to bind to its intended antigenic determinant. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the Nanobodies of theinvention). For example, linkers containing one or more charged aminoacid residues (see Table A-2 above) can provide improved hydrophilicproperties, whereas linkers that form or contain small epitopes or tagscan be used for the purposes of detection, identification and/orpurification. Again, based on the disclosure herein, the skilled personwill be able to determine the optimal linkers for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thereto. For example, when a polypeptideof the invention comprises three of more Nanobodies, it is possible tolink them by use of a linker with three or more “arms”, which each “arm”being linked to a Nanobody, so as to provide a “star-shaped” construct.It is also possible, although usually less preferred, to use circularconstructs.

The invention also comprises derivatives of the polypeptides of theinvention, which may be essentially analogous to the derivatives of theNanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that “essentiallyconsist” of a polypeptide of the invention (in which the wording“essentially consist of” has essentially the same meaning as indicatedhereinabove).

According to one aspect of the invention, the polypeptide of theinvention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids ofthe invention can be prepared in a manner known per se, as will be clearto the skilled person from the further description herein. For example,the Nanobodies and polypeptides of the invention can be prepared in anymanner known per se for the preparation of antibodies and in particularfor the preparation of antibody fragments (including but not limited to(single) domain antibodies and ScFv fragments). Some preferred, butnon-limiting methods for preparing the amino acid sequences, Nanobodies,polypeptides and nucleic acids include the methods and techniquesdescribed herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, Nanobody and/or a polypeptide ofthe invention generally comprises the steps of:

-   i) the expression, in a suitable host cell or host organism (also    referred to herein as a “host of the invention”) or in another    suitable expression system of a nucleic acid that encodes said amino    acid sequence, Nanobody or polypeptide of the invention (also    referred to herein as a “nucleic acid of the invention”), optionally    followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under    conditions that are such that said host of the invention expresses    and/or produces at least one amino acid sequence, Nanobody and/or    polypeptide of the invention; optionally followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can forexample be subjected to site-directed mutagenesis, so at to provide anucleic acid of the invention encoding said analog. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding a Nanobody and for example nucleic acids encoding oneor more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring form of a metalloproteinase from the ADAM family asa template. These and other techniques will be clear to the skilledperson, and reference is again made to the standard handbooks, such asSambrook et al. and Ausubel et al., mentioned above, as well as theExamples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting aspect, a genetic construct of theinvention comprises

-   i) at least one nucleic acid of the invention; operably connected to-   ii) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;    and optionally also-   iii) one or more further elements of genetic constructs known per    se;    in which the terms “regulatory element”, “promoter”, “terminator”    and “operably connected” have their usual meaning in the art (as    further described herein); and in which said “further elements”    present in the genetic constructs may for example be 3′- or 5′-UTR    sequences, leader sequences, selection markers, expression    markers/reporter genes, and/or elements that may facilitate or    increase (the efficiency of) transformation or integration. These    and other suitable elements for such genetic constructs will be    clear to the skilled person, and may for instance depend upon the    type of construct used, the intended host cell or host organism; the    manner in which the nucleotide sequences of the invention of    interest are to be expressed (e.g. via constitutive, transient or    inducible expression); and/or the transformation technique to be    used. For example, regulatory sequences, promoters and terminators    known per se for the expression and production of antibodies and    antibody fragments (including but not limited to (single) domain    antibodies and ScFv fragments) may be used in an essentially    analogous manner.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promoter). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

Some particularly preferred promoters include, but are not limited to,promoters known per se for the expression in the host cells mentionedherein; and in particular promoters for the expression in the bacterialcells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriateselection conditions—host cells and/or host organisms that have been(successfully) transformed with the nucleotide sequence of the inventionto be distinguished from host cells/organisms that have not been(successfully) transformed. Some preferred, but non-limiting examples ofsuch markers are genes that provide resistance against antibiotics (suchas kanamycin or ampicillin), genes that provide for temperatureresistance, or genes that allow the host cell or host organism to bemaintained in the absence of certain factors, compounds and/or (food)components in the medium that are essential for survival of thenon-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or hostorganism—it allows for the desired post-translational modificationsand/or such that it directs the transcribed mRNA to a desired part ororganelle of a cell. A leader sequence may also allow for secretion ofthe expression product from said cell. As such, the leader sequence maybe any pro-, pre-, or prepro-sequence operable in the host cell or hostorganism. Leader sequences may not be required for expression in abacterial cell. For example, leader sequences known per se for theexpression and production of antibodies and antibody fragments(including but not limited to single domain antibodies and ScFvfragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that—in the hostcell or host organism—it allows for detection of the expression of (agene or nucleotide sequence present on) the genetic construct. Anexpression marker may optionally also allow for the localisation of theexpressed product, e.g. in a specific part or organelle of a cell and/orin (a) specific cell(s), tissue(s), organ(s) or part(s) of amulticellular organism. Such reporter genes may also be expressed as aprotein fusion with the amino acid sequence of the invention. Somepreferred, but non-limiting examples include fluorescent proteins suchas GFP.

Some preferred, but non-limiting examples of suitable promoters,terminator and further elements include those that can be used for theexpression in the host cells mentioned herein; and in particular thosethat are suitable for expression in bacterial cells, such as thosementioned herein and/or those used in the Examples below. For some(further) non-limiting examples of the promoters, selection markers,leader sequences, expression markers and further elements that may bepresent/used in the genetic constructs of the invention—such asterminators, transcriptional and/or translational enhancers and/orintegration factors—reference is made to the general handbooks such asSambrook et al. and Ausubel et al. mentioned above, as well as to theexamples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat.No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1 085 089. Other exampleswill be clear to the skilled person. Reference is also made to thegeneral background art cited above and the further references citedherein.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the amino acid sequence, Nanobody orpolypeptide of the invention. Suitable hosts or host cells will be clearto the skilled person, and may for example be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        lividans; of Staphylococcus, for example of Staphylococcus        carnosus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosaccharomyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorpha;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for example of Arxula adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example a cell or cell line        derived from a human, a cell or a cell line from mammals        including but not limited to CHO-cells, BHK-cells (for example        BHK-21 cells) and human cells or cell lines such as HeLa, COS        (for example COS-7) and PER.C6 cells;        as well as all other hosts or host cells known per se for the        expression and production of antibodies and antibody fragments        (including but not limited to (single) domain antibodies and        ScFv fragments), which will be clear to the skilled person.        Reference is also made to the general background art cited        hereinabove, as well as to for example WO 94/29457; WO 96/34103;        WO 99/42077; Frenken et al., (1998), supra; Riechmann and        Muyldermans, (1999), supra; van der Linden, (2000), supra;        Thomassen et al., (2002), supra; Joosten et al., (2003), supra;        Joosten et al., (2005), supra; and the further references cited        herein.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be introduced and expressed in one or more cells, tissues ororgans of a multicellular organism, for example for prophylactic and/ortherapeutic purposes (e.g. as a gene therapy). For this purpose, thenucleotide sequences of the invention may be introduced into the cellsor tissues in any suitable way, for example as such (e.g. usingliposomes) or after they have been inserted into a suitable gene therapyvector (for example derived from retroviruses such as adenovirus, orparvoviruses such as adeno-associated virus). As will also be clear tothe skilled person, such gene therapy may be performed in vivo and/or insitu in the body of a patient by administering a nucleic acid of theinvention or a suitable gene therapy vector encoding the same to thepatient or to specific cells or a specific tissue or organ of thepatient; or suitable cells (often taken from the body of the patient tobe treated, such as explanted lymphocytes, bone marrow aspirates ortissue biopsies) may be treated in vitro with a nucleotide sequence ofthe invention and then be suitably (re-)introduced into the body of thepatient. All this can be performed using gene therapy vectors,techniques and delivery systems which are well known to the skilledperson, and for example described in Culver, K. W., “Gene Therapy”,1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.);Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79(1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, GeneTher. 5 (1998), 692-699; Nabel, Ann N.Y. Acad. Sci.: 811 (1997),289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, NatureMedicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat. No.5,580,859; U.S. Pat. No. 55,895,466; or Schaper, Current Opinion inBiotechnology 7 (1996), 635-640. For example, in situ expression of ScFvfragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and ofdiabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has beendescribed in the art.

For expression of the Nanobodies in a cell, they may also be expressedas so-called “intrabodies”, as for example described in WO 94/02610, WO95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. &Biocca, S. (1997) Intracellular Antibodies: Development andApplications. Landes and Springer-Verlag; and in Kontermann, Methods 34,(2004), 163-170.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan for example also be produced in the milk of transgenic mammals, forexample in the milk of rabbits, cows, goats or sheep (see for exampleU.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No.6,849,992 for general techniques for introducing transgenes intomammals), in plants or parts of plants including but not limited totheir leaves, flowers, fruits, seed, roots or tubers (for example intobacco, maize, soybean or alfalfa) or in for example pupae of thesilkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and polypeptides ofthe invention can also be expressed and/or produced in cell-freeexpression systems, and suitable examples of such systems will be clearto the skilled person. Some preferred, but non-limiting examples includeexpression in the wheat germ system; in rabbit reticulocyte lysates; orin the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies isthat the polypeptides based thereon can be prepared through expressionin a suitable bacterial system, and suitable bacterial expressionsystems, vectors, host cells, regulatory elements, etc., will be clearto the skilled person, for example from the references cited above. Itshould however be noted that the invention in its broadest sense is notlimited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of Nanobodies or Nanobody-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceutical (i.e. GMP grade)expression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a Nanobody-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast usually leads to a glycosylation pattern that differs fromhuman glycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, Nanobody orpolypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention is glycosylated.According to another non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention isnon-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the amino acid sequence, Nanobody or polypeptide of the invention isproduced in a bacterial cell, in particular a bacterial cell suitablefor large scale pharmaceutical production, such as cells of the strainsmentioned above.

According to another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned above.

According to yet another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acidsequences, Nanobodies and the polypeptides of the invention, the aminoacid sequences, Nanobodies and polypeptides of the invention can beproduced either intracellullarly (e.g. in the cytosol, in the periplasmaor in inclusion bodies) and then isolated from the host cells andoptionally further purified; or can be produced extracellularly (e.g. inthe medium in which the host cells are cultured) and then isolated fromthe culture medium and optionally further purified. When eukaryotic hostcells are used, extracellular production is usually preferred since thisconsiderably facilitates the further isolation and downstream processingof the Nanobodies and proteins obtained. Bacterial cells such as thestrains of E. coli mentioned above normally do not secrete proteinsextracellularly, except for a few classes of proteins such as toxins andhemolysin, and secretory production in E. coli refers to thetranslocation of proteins across the inner membrane to the periplasmicspace. Periplasmic production provides several advantages over cytosolicproduction. For example, the N-terminal amino acid sequence of thesecreted product can be identical to the natural gene product aftercleavage of the secretion signal sequence by a specific signalpeptidase. Also, there appears to be much less protease activity in theperiplasm than in the cytoplasm. In addition, protein purification issimpler due to fewer contaminating proteins in the periplasm. Anotheradvantage is that correct disulfide bonds may form because the periplasmprovides a more oxidative environment than the cytoplasm. Proteinsoverexpressed in E. coli are often found in insoluble aggregates,so-called inclusion bodies. These inclusion bodies may be located in thecytosol or in the periplasm; the recovery of biologically activeproteins from these inclusion bodies requires a denaturation/refoldingprocess. Many recombinant proteins, including therapeutic proteins, arerecovered from inclusion bodies. Alternatively, as will be clear to theskilled person, recombinant strains of bacteria that have beengenetically modified so as to secrete a desired protein, and inparticular an amino acid sequence, Nanobody or a polypeptide of theinvention, can be used.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention is an amino acidsequence, Nanobody or polypeptide that has been produced intracellularlyand that has been isolated from the host cell, and in particular from abacterial cell or from an inclusion body in a bacterial cell. Accordingto another non-limiting aspect of the invention, the amino acidsequence, Nanobody or polypeptide of the invention is an amino acidsequence, Nanobody or polypeptide that has been producedextracellularly, and that has been isolated from the medium in which thehost cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude,

-   -   for expression in E. coli: lac promoter (and derivatives thereof        such as the lacUV5 promoter); arabinose promoter; left-(PL) and        rightward (PR) promoter of phage lambda; promoter of the trp        operon; hybrid lac/trp promoters (tac and trc); T7-promoter        (more specifically that of T7-phage gene 10) and other T-phage        promoters; promoter of the Tn10 tetracycline resistance gene;        engineered variants of the above promoters that include one or        more copies of an extraneous regulatory operator sequence;    -   for expression in S. cerevisiae: constitutive: ADH1 (alcohol        dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),        GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1        (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:        GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol        dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper        metallothionein); heterologous: CaMV (cauliflower mosaic virus        35S promoter);    -   for expression in Pichia pastoris: the AOX1 promoter (alcohol        oxidase I);    -   for expression in mammalian cells: human cytomegalovirus (hCMV)        immediate early enhancer/promoter; human cytomegalovirus (hCMV)        immediate early promoter variant that contains two tetracycline        operator sequences such that the promoter can be regulated by        the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)        promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)        enhancer/promoter; elongation factor 1α (hEF-1α) promoter from        human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1        long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cellsinclude:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),        pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene),        EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),        pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo        (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and        1ZD35 (ATCC 37565), as well as viral-based expression systems,        such as those based on adenovirus;    -   vectors for expression in bacterial cells: pET vectors (Novagen)        and pQE vectors (Qiagen);    -   vectors for expression in yeast or other fungal cells: pYES2        (Invitrogen) and Pichia expression vectors (Invitrogen);    -   vectors for expression in insect cells: pBlueBacII (Invitrogen)        and other baculovirus vectors    -   vectors for expression in plants or plant cells: for example        vectors based on cauliflower mosaic virus or tobacco mosaic        virus, suitable strains of Agrobacterium, or Ti-plasmid based        vectors.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include:

-   -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,        OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the        like; TAT signal peptide, hemolysin C-terminal secretion signal;    -   for use in yeast: α-mating factor prepro-sequence, phosphatase        (pho1), invertase (Suc), etc.;    -   for use in mammalian cells: indigenous signal in case the target        protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal        peptide; etc.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence, Nanobody or polypeptide of theinvention (and in case of a host organism: in at least one cell, part,tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence, Nanobody or polypeptide of theinvention is expressed/produced. Suitable conditions will be clear tothe skilled person and will usually depend upon the host cell/hostorganism used, as well as on the regulatory elements that control theexpression of the (relevant) nucleotide sequence of the invention.Again, reference is made to the handbooks and patent applicationsmentioned above in the paragraphs on the genetic constructs of theinvention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acidsequence, Nanobody or polypeptide of the invention may (first) begenerated in an immature form (as mentioned above), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, Nanobody orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention maythen be isolated from the host cell/host organism and/or from the mediumin which said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, Nanobody or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or compositions comprisingat least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one Nanobody of the invention or at least one polypeptide of theinvention and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances.

Generally, the amino acid sequences, Nanobodies and polypeptides of theinvention can be formulated and administered in any suitable mannerknown per se, for which reference is for example made to the generalbackground art cited above (and in particular to WO 04/041862, WO04/041863, WO 04/041865 and WO 04/041867) as well as to the standardhandbooks, such as Remington's Pharmaceutical Sciences, 18^(th) Ed.,Mack Publishing Company, USA (1990) or Remington, the Science andPractice of Pharmacy, 21st Edition, Lippincott Williams and Wilkins(2005).

For example, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be formulated and administered in any manner known perse for conventional antibodies and antibody fragments (including ScFv'sand diabodies) and other pharmaceutically active proteins. Suchformulations and methods for preparing the same will be clear to theskilled person, and for example include preparations suitable forparenteral administration (for example intravenous, intraperitoneal,subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecaladministration) or for topical (i.e. transdermal or intradermal)administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andaqueous buffers and solutions such as physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution;water oils; glycerol; ethanol; glycols such as propylene glycol or aswell as mineral oils, animal oils and vegetable oils, for example peanutoil, soybean oil, as well as suitable mixtures thereof. Usually, aqueoussolutions or suspensions will be preferred.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be administered using gene therapy methods of delivery. See,e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in itsentirety. Using a gene therapy method of delivery, primary cellstransfected with the gene encoding an amino acid sequence, Nanobody orpolypeptide of the invention can additionally be transfected with tissuespecific promoters to target specific organs, tissue, grafts, tumors, orcells and can additionally be transfected with signal and stabilizationsequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies and polypeptides of theinvention may be systemically administered, e.g., orally, in combinationwith a pharmaceutically acceptable vehicle such as an inert diluent oran assimilable edible carrier. They may be enclosed in hard or softshell gelatin capsules, may be compressed into tablets, or may beincorporated directly with the food of the patient's diet. For oraltherapeutic administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of theamino acid sequence, Nanobody or polypeptide of the invention. Theirpercentage in the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the amino acidsequence, Nanobody or polypeptide of the invention in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the amino acid sequences, Nanobodies and polypeptides of theinvention, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the amino acid sequences,Nanobodies and polypeptides of the invention may be incorporated intosustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be administered intravenously or intraperitoneally by infusionor injection. Solutions of the amino acid sequences, Nanobodies andpolypeptides of the invention or their salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the aminoacid sequences, Nanobodies and polypeptides of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be applied in pure form, i.e., whenthey are liquids. However, it will generally be desirable to administerthem to the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the amino acid sequences, Nanobodies and polypeptidesof the invention can be dissolved or dispersed at effective levels,optionally with the aid of non-toxic surfactants. Adjuvants such asfragrances and additional antimicrobial agents can be added to optimizethe properties for a given use. The resultant liquid compositions can beapplied from absorbent pads, used to impregnate bandages and otherdressings, or sprayed onto the affected area using pump-type or aerosolsprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the amino acid sequences, Nanobodies and polypeptides of theinvention to the skin are known to the art; for example, see Jacquet etal. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith etal. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, Nanobodies and polypeptidesof the invention can be determined by comparing their in vitro activity,and in vivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequences, Nanobodies andpolypeptides of the invention in a liquid composition, such as a lotion,will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. Theconcentration in a semi-solid or solid composition such as a gel or apowder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the amino acid sequences, Nanobodies and polypeptides ofthe invention required for use in treatment will vary not only with theparticular amino acid sequence, Nanobody or polypeptide selected butalso with the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician. Also thedosage of the amino acid sequences, Nanobodies and polypeptides of theinvention varies depending on the target cell, tumor, tissue, graft, ororgan.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one ADAM-related diseases and disorders,said method comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with ametalloproteinase from the ADAM family, with its biological orpharmacological activity, and/or with the biological pathways orsignaling in which a metalloproteinase from the ADAM family is involved,said method comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can be treatedby modulating a metalloproteinase from the ADAM family, its biologicalor pharmacological activity, and/or the biological pathways or signalingin which a metalloproteinase from the ADAM family is involved, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, said pharmaceutically effective amount may be an amountthat is sufficient to modulate a metalloproteinase from the ADAM family,its biological or pharmacological activity, and/or the biologicalpathways or signaling in which a metalloproteinase from the ADAM familyis involved; and/or an amount that provides a level of the amino acidsequence of the invention, of a Nanobody of the invention, of apolypeptide of the invention in the circulation that is sufficient tomodulate a metalloproteinase from the ADAM family, its biological orpharmacological activity, and/or the biological pathways or signaling inwhich a metalloproteinase from the ADAM family is involved.

The invention furthermore relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering an amino acid sequence of the invention,a Nanobody of the invention or a polypeptide of the invention to apatient, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of anamino acid sequence of the invention, of a Nanobody of the invention, ofa polypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In the above methods, the amino acid sequences, Nanobodies and/orpolypeptides of the invention and/or the compositions comprising thesame can be administered in any suitable manner, depending on thespecific pharmaceutical formulation or composition to be used. Thus, theamino acid sequences, Nanobodies and/or polypeptides of the inventionand/or the compositions comprising the same can for example beadministered orally, intraperitoneally (e.g. intravenously,subcutaneously, intramuscularly, or via any other route ofadministration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The amino acid sequences, Nanobodies and/or polypeptides of theinvention and/or the compositions comprising the same are administeredaccording to a regime of treatment that is suitable for preventingand/or treating the disease or disorder to be prevented or treated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease or disorder to beprevented or treated, the severity of the disease to be treated and/orthe severity of the symptoms thereof, the specific amino acid sequence,Nanobody or polypeptide of the invention to be used, the specific routeof administration and pharmaceutical formulation or composition to beused, the age, gender, weight, diet, general condition of the patient,and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, Nanobodies and/or polypeptides of theinvention, or of one or more compositions comprising the same, in one ormore pharmaceutically effective amounts or doses. The specific amount(s)or doses to administered can be determined by the clinician, again basedon the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,Nanobody and polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the amino acid sequences, Nanobodies and polypeptidesof the invention will generally be administered in an amount between 1gram and 0.01 microgram per kg body weight per day, preferably between0.1 gram and 0.1 microgram per kg body weight per day, such as about 1,10, 100 or 1000 microgram per kg body weight per day, eithercontinuously (e.g. by infusion), as a single daily dose or as multipledivided doses during the day. The clinician will generally be able todetermine a suitable daily dose, depending on the factors mentionedherein. It will also be clear that in specific cases, the clinician maychoose to deviate from these amounts, for example on the basis of thefactors cited above and his expert judgment. Generally, some guidance onthe amounts to be administered can be obtained from the amounts usuallyadministered for comparable conventional antibodies or antibodyfragments against the same target administered via essentially the sameroute, taking into account however differences in affinity/avidity,efficacy, biodistribution, half-life and similar factors well known tothe skilled person.

Usually, in the above method, a single amino acid sequence, Nanobody orpolypeptide of the invention will be used. It is however within thescope of the invention to use two or more amino acid sequences,Nanobodies and/or polypeptides of the invention in combination.

The Nanobodies, amino acid sequences and polypeptides of the inventionmay also be used in combination with one or more furtherpharmaceutically active compounds or principles, i.e. as a combinedtreatment regimen, which may or may not lead to a synergistic effect.Again, the clinician will be able to select such further compounds orprinciples, as well as a suitable combined treatment regimen, based onthe factors cited above and his expert judgement.

In particular, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be used in combination with other pharmaceuticallyactive compounds or principles that are or can be used for theprevention and/or treatment of the diseases and disorders cited herein,as a result of which a synergistic effect may or may not be obtained.Examples of such compounds and principles, as well as routes, methodsand pharmaceutical formulations or compositions for administering themwill be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for prevention and/or treatment of at leastone ADAM-related diseases and disorders; and/or for use in one or moreof the methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention in the preparation of apharmaceutical composition for the prevention and/or treatment of atleast one disease or disorder that can be prevented and/or treated byadministering an amino acid sequence, Nanobody or polypeptide of theinvention to a patient.

More in particular, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for the prevention and/or treatment ofADAM-related diseases and disorders, and in particular for theprevention and treatment of one or more of the diseases and disorderslisted herein.

Again, in such a pharmaceutical composition, the one or more amino acidsequences, Nanobodies or polypeptides of the invention may also besuitably combined with one or more other active principles, such asthose mentioned herein.

Finally, although the use of the Nanobodies of the invention (as definedherein) and of the polypeptides of the invention is much preferred, itwill be clear that on the basis of the description herein, the skilledperson will also be able to design and/or generate, in an analogousmanner, other amino acid sequences and in particular (single) domainantibodies against a metalloproteinase from the ADAM family, as well aspolypeptides comprising such (single) domain antibodies.

For example, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNanobodies of the invention onto such (single) domain antibodies orother protein scaffolds, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting will be clear to the skilled person and are well known inthe art, see for example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605522, EP 0 460 167, U.S. Pat. No. 7,054,297, Nicaise et al., ProteinScience (2004), 13:1882-1891; Ewert et al., Methods, 2004 October;34(2):184-199; Kettleborough et al., Protein Eng. 1991 October; 4(7):773-783; O'Brien and Jones, Methods Mol. Biol. 2003: 207: 81-100;Skerra, J. Mol. Recognit. 2000: 13: 167-187, and Saerens et al., J. Mol.Biol. 2005 Sep. 23; 352(3):597-607, and the further references citedtherein. For example, techniques known per se for grafting mouse or ratCDR's onto human frameworks and scaffolds can be used in an analogousmanner to provide chimeric proteins comprising one or more of the CDR'sof the Nanobodies of the invention and one or more human frameworkregions or sequences.

It should also be noted that, when the Nanobodies of the inventionscontain one or more other CDR sequences than the preferred CDR sequencesmentioned above, these CDR sequences can be obtained in any manner knownper se, for example from Nanobodies (preferred), V_(H) domains fromconventional antibodies (and in particular from human antibodies), heavychain antibodies, conventional 4-chain antibodies (such as conventionalhuman 4-chain antibodies) or other immunoglobulin sequences directedagainst a metalloproteinase from the ADAM family. Such immunoglobulinsequences directed against a metalloproteinase from the ADAM family canbe generated in any manner known per se, as will be clear to the skilledperson, i.e. by immunization with a metalloproteinase from the ADAMfamily or by screening a suitable library of immunoglobulin sequenceswith a metalloproteinase from the ADAM family, or any suitablecombination thereof. Optionally, this may be followed by techniques suchas random or site-directed mutagenesis and/or other techniques foraffinity maturation known per se. Suitable techniques for generatingsuch immunoglobulin sequences will be clear to the skilled person, andfor example include the screening techniques reviewed by Hoogenboom,Nature Biotechnology, 23, 9, 1105-1116 (2005) Other techniques forgenerating immunoglobulins against a specified target include forexample the Nanoclone technology (as for example described in thepublished US patent application 2006-0211088), so-called SLAM technology(as for example described in the European patent application 0 542 810),the use of transgenic mice expressing human immunoglobulins or thewell-known hybridoma techniques (see for example Larrick et al,Biotechnology, Vol. 7, 1989, p. 934). All these techniques can be usedto generate immunoglobulins against a metalloproteinase from the ADAMfamily, and the CDR's of such immunoglobulins can be used in theNanobodies of the invention, i.e. as outlined above. For example, thesequence of such a CDR can be determined, synthesized and/or isolated,and inserted into the sequence of a Nanobody of the invention (e.g. soas to replace the corresponding native CDR), all using techniques knownper se such as those described herein, or Nanobodies of the inventioncontaining such CDR's (or nucleic acids encoding the same) can besynthesized de novo, again using the techniques mentioned herein.

Further uses of the amino acid sequences, Nanobodies, polypeptides,nucleic acids, genetic constructs and hosts and host cells of theinvention will be clear to the skilled person based on the disclosureherein. For example, and without limitation, the amino acid sequences ofthe invention can be linked to a suitable carrier or solid support so asto provide a medium than can be used in a manner known per se to purifya metalloproteinase from the ADAM family from compositions andpreparations comprising the same. Derivatives of the amino acidsequences of the invention that comprise a suitable detectable label canalso be used as markers to determine (qualitatively or quantitatively)the presence of a metalloproteinase from the ADAM family in acomposition or preparation or as a marker to selectively detect thepresence of a metalloproteinase from the ADAM family on the surface of acell or tissue (for example, in combination with suitable cell sortingtechniques).

The invention will now be further described by means of the followingnon-limiting Experimental Part and the non-limiting Figures, in which:

FIG. 1 is a graph showing the effect of purified nanobodies (1 uM, 25molar excess) on the activity of recombinant ADAM9 (150 ng=40 nM, R&Dsystem).

FIG. 2 is a graph showing the effect of increasing amount of purifiednanobodies [2× (16 nM) to 50× (400 nM) excess relative to ADAM17) on theactivity of recombinant ADAM17 (40 ng=8 nM, R&D system).

FIG. 3 is a similar graph to FIG. 2, showing the effect of increasedNanobody concentration on ADAM17 activity.

FIG. 4 is a graph showing the effect of purified nanobodies (400 nM, 10molar excess) on the activity of recombinant ADAM10 (200 ng=40 nM, R&Dsystem).

FIG. 5 is a graph showing Effect of the increasing amount of purifiednanobodies (excess relative to ADAM10) on the activity of recombinantADAM10 (R&D system).

FIG. 6 is a graph showing the effect of 50 molar excess of purifiednanobodies on the activity of recombinant ADAM10 (R&D system). On top ofeach column is indicated the percent residual activity.

EXPERIMENTAL PART Example 1 Immunizations

Two llamas (131 and 132) were immunized according to standard protocolswith 6 boosts of R&D Systems Cat #2717-PG and a cocktail of ADAM(recombinant human ADAM8 (amino residues 1-497, R&D Systems, Cat. No.1031-AD, Lot No. MWP04607A), recombinant human ADAM9 (amino residues1-697, R&D Systems, Cat. No. 939-AD, Lot No. FQO03607A), recombinanthuman ADAM10 (amino residues 18-672, Cat. No 936-AD, Lot No: FDW12607A),recombinant human ADAM17/TACE (amino residues 1-671, Cat. No. 930-ADB,Lot No. GDU12607A) and recombinant human ADAMTS5 (amino residues 1-622,Cat. No. 2198-AD, Lot No OLY02607A). Blood was collected from theseanimals after 7 days after boost 6 and 10 days after boost 6.

Example 2 Library Construction

Peripheral blood mononuclear cells were prepared from blood samplesusing Ficoll-Hypaque according to the manufacturer's instructions. Next,total RNA extracted was extracted from these cells and used as startingmaterial for RT-PCR to amplify Nanobody encoding gene fragments. Thesefragments were cloned into phagemid vector pAX50. Phage was preparedaccording to standard methods (see for example the prior art andapplications filed by applicant cited herein) and stored at 4° C. forfurther use.

Example 3 Selections

Phage libraries 131 and 132 were used for selections on the ADAMs thatwere used for immunization. The ADAMs were coated individually at 5ug/ml, 0.5 ug/ml or 0 ug/ml (control) on Nunc Maxisorp ELISA plates.Binding phages were eluted from the coated ADAMs with Trypsine (R1).

Output of both R1 selections were analyzed for enrichment factor (phagepresent in eluate relative to controls). Based on these parameters thebest selections were chosen for further analysis. When only few positiveclones were recovered, a second round of selection was done using thesame condition as for the first round of selection and trypsine elution(R2).

The Nanobody encoding fragments from R1 and R2 were recloned into theplasmid pAX51 according to standard methods and individual colonies werepicked up and grown in 96 deep well plates (1 ml volume) and induced byadding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80ul) were prepared according to standard methods (see for example theprior art and applications filed by applicant cited herein).

Example 4 Screening for Binding

In order to determine the binding specificity of the Nanobodies to theADAMs, the clones were tested in an ELISA assay.

In short, 1 ug/ml of indicated ADAM were immobilized directly onMaxisorp microtiter plates (Nunc). Free binding sites were blocked using4% Marvel in PBS. Next, 5 ul of periplasmic extract containing Nanobodyof the different clones in 100 ul 2% Marvel PBST were allowed to bind tothe immobilized antigen. After incubation and a wash step, Nanobodybinding was revealed using a mouse-anti-myc secondary antibody, whichwas after a wash step detected with a HRP-conjugated goat-anti-mouseantibody. Binding specificity was determined based on OD values comparedto controls having received no Nanobody (low control, FIG. 1 for R1 andFIG. 2 for R2)

TABLE B-1 Positive clones identify by ELISA for each ADAM after thefirst round of selection (left table) and second round of selection(right table). R1 R2 LIB 131 LIB 132 TOTAL LIB 131 LIB 132 TOTAL (% pos)(% pos) (% pos) (% pos) (% pos) (% pos) ADAM8 17 (77%)  8 (36%) 25 (57%)ADAM8 ADAM9 12 (55%)  6 (27%) 18 (41%) ADAM9 13 (28%) 13 (28%) ADAM10  6(27%) 2 (9%)  8 (18%) ADAM10 43 (93%) 24 (52%) 67 (73%) ADAM17 0 (0%) 10(45%) 10 (23%) ADAM17 43 (93%) 43 (93%) ADAMTS5  6 (27%) 14 (64%) 20(45%) ADAMTS5 28 (61%) 28 (61%) Are depicted the number of positiveidentified as well as the representative percentage of positive.

Example 5 Screening for Activity

In order to determine the effect of the Nanobodies on the proteaseactivity, we used an available commercial fluorescent peptide substrateto do an ADAM protease assay (R&D system, cat ES003). Shortly, theperiplasms containing the Nanobodies (dialysed against Tris pH9) werepreincubated with the ADAM protein for 1 h. Next, the fluorogenicpeptide substrate (R&D system, cat ES003) was added and the cleavedproduct detected in time at 25 C according to manufacturer instruction.

Out of 25 periplasm tested for ADAM17, 6 were inhibiting (less than 80%residual activity) and 6 activating (more than 120% initial activity)compared to other periplasms.

Out of 27 periplasm tested for ADAM10, 5 inhibited significantly (lessthan 40% residual activity) and 6 activated significantly (more than200% initial activity) compared to other periplasms.

Out of 31 periplasm tested for ADAM9, 7 showed significant inhibitionand 9 were significantly activating.

To confirm the effect of the nanobodies against the ADAMs, thenanobodies were purified, dialysed against 20 nM tris pH9 to avoid thepresence of inactivating salts.

The results for the nanobodies against ADAM9 are shown in FIG. 1, whichschematically shows the effect of purified nanobodies (1 uM, 25 molarexcess) on the activity of recombinant ADAM9 (150 ng=40 nM, R&D system).Because of the somehow stabilizing effect that the presence of nonrelevant protein has on the ADAM9, the average activity was setupbetween 110 and 160 fluorescent unit (FU). As can be seen from FIG. 1,nanobodies 30F2, 30D1, 30B6 (also 30E12, but not shown here) have aninactivating effect and Nanobodies 41B5, 30E6, 30C1, 31E12 and 30G12have an activating effect.

A similar experiment was performed in respect of ADAM17. The results areschematically shown in FIGS. 2 and 3, from which can be seen thatnanobodies 35B1, 35C12, 32E1, 40A6 have an inactivating effect andnanobodies 35A12, 34H1 and 33G6 have an activating effect. The strikingstabilizing effect that the presence of non-relevant protein has on theADAM17 (see non-relevant 11F2 nanobody at 25× excess) should be noted.

A similar experiment was performed in respect of ADAM10. The results areschematically shown in FIG. 4, which shows the effect of purifiednanobodies (400 nM, 10 molar excess) on the activity of recombinantADAM10 (200 ng=40 nM, R&D system). As can be seen from FIG. 4,nanobodies 39D8, 39B1, 39D1 and 33D1 have an inactivating effect, andnanobodies 39F1, 32E1 (have related sequence=family 2) and 33G6, 39G8,39H8, 39A8 and 39C8 (all part of the same family 1) have an activatingeffect. The experiment was repeated with some clones, with the resultsbeing schematically shown in FIGS. 5 and 6, which shows the effect ofthe increasing amount of purified nanobodies (excess relative to ADAM10)on the activity of recombinant ADAM10 (R&D system). As can be seen fromFIG. 5, nanobodies 39D8, 39B1, 39D1 and 33D1 have an inactivatingeffect, and nanobodies 39C8, 32E1 and 39F1 have an activating effect.The controls (11F2 and 30E6) showed little to no effect (see also FIG.6, which shows the effect of 50 molar excess of purified nanobodies onthe activity of recombinant ADAM10 (R&D system).

The activity of amino acid sequences or polypeptides of the invention onADAM-TS5 can be tested using an commercially available ELISA-based kit(from Invitek). For testing the activity of amino acid sequences orpolypeptides of the invention on ADAM10 in a cell-based assay, cleavageof CD44 into the medium of cell can be used to detect the activity ofADAM10 in U251 glioblastoma (S. Atkinson and G. Murphy, publicationsubmitted). For testing the activity of amino acid sequences orpolypeptides of the invention on ADAM17 in a cell-based assay, therelease of HB-EGF conjugated Alkaline phosphatase from transfected MCF7cells can be used a readout for ADAM17 activity (S. Atkinson and G.Murphy, submitted).

For testing the activity of amino acid sequences or polypeptides of theinvention on ADAM10 in a cell-based assay, cleavage of CD44 into themedium of cell can be used to detect the activity of ADAM10 in U251glioblastoma (S. Atkinson and G. Murphy, publication submitted). Fortesting the activity of amino acid sequences or polypeptides of theinvention on ADAM17 in a cell-based assay, the release of HB-EGFconjugated Alkaline phosphatase from transfected MCF7 cells can be useda readout for ADAM17 activity (S. Atkinson and G. Murphy, submitted).

Table B-2 gives the amino acid sequences of a number of Nanobodiesagainst some representative ADAM proteinases.

TABLE B-2 Nanobodies against some representative ADAMmetalloproteinases. SEQ ID NO's 868 to 886: Nanobodies against ADAM8 SEQID NO's 887 to 907 and 1044 to 1047: Nanobodies against ADAM9 SEQ IDNO's 908 to 931: Nanobodies against ADAM10 SEQ ID NO's 932 to 948 and1048 to 1052: Nanobodies against ADAM17 SEQ ID NO's 949 to 974 and 1053:Nanobodies against ADAMTS5 >28-B12, C12, D6, E12 SEQ ID NO: 868EVQLVESGGGLVQNGGSLRLSCVMSGSSISLYTSGWYRQAPGKQREWVASIASGASGGTTVYEDSVKGRFTISRDDAENTVYLQMITLKPEDTAVYYCRAQDPMRTRDAYWGQGTQVTVSS >28-D1, H1 SEQ ID NO: 869EVQLVESGGGLVQAGGSLRLSCAASGRTFDNYIMGWFRQAPGKEREFVAAINYEGDRTYSPNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYQCATGPRYGKYDYWGQGTQVTVSS >28-A6 SEQ ID NO: 870EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYTMYWFRQAPGKERELVAAIGGVTDYADSVKGRFTIARDNAKSTVSLQMNSLKPEDTAVYYCAAKHRSVATRTGGNVSWGQGTQVTVSS >28-B6 SEQ ID NO: 871EVQLVESGGGLVQAGASLKISCAASGRTYDIRVMGWFRRAPGKDRESVATVTWRDNITYYVDSVKGRFTITRDNAKNTVYLQMNNLKPEDTAVYYCAAQTEDSAQYIYWGQGTQVTVSS >28-C1 SEQ ID NO: 872EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYVMGWFRQAPGREREFVAAMGDTTLYADSVEGRFTISRDNDQNTVYLQMSSLKPEDTAAYFCAARSRFIPPLTFRTGGAYDYWGQGTQVTVSS >28-C6 SEQ ID NO: 873EVQLVESGGGLVQAGASLKLSCAASGRTYDIRVMGWFRRAPGKGRESVATTTWRDNITYYMDSVKGRFTITRDNAKNTVYLQMNNLKPEDTAVYYCAAQTEDSAQYIYWGQGTQVTVSS >28-D12 SEQ ID NO: 874EVQLVESGGGLVQAGGSLRLSCATSGSIASFNAMAWHRQAPGNQRELVAAIHISGNTNYADSVKGRFTISRDNGKNTVYLQMNSLRPEDTAVYYCNAIEVRTGLSPRGHWGQGTQVTVSS >28-E1 SEQ ID NO: 875EVQLVESGGGLVQAGGSLRLSCTASGRTFSDYTMAWFRQAPGKERELVAAIGSVTDYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCAAKHRTRTRGYVNWGQGTQVTVSS >28-E6 SEQ ID NO: 876EVQLVESGGGLVQAGGSLRLSCAASGNIFRIETMAWHRQAPGKQRELVAAIRSDDMTNYPDSVKGRFTISRDNFKNTVYLQMNSLTPEDTAVYYCNLIQRRAPYSRLETYWGQGTQVTVSS >28-F12 SEQ ID NO: 877EVQLVESGGGLVQAGESLRLSCKASRNDFSFNSMAWYRQSPGKQRNLVARIFRSANIYYDDSVKGRFTISRDSALNTVFLQMNALKSEDTAVYYCNGRLSNGLDYWGQGTQVTVSS >28-G1 SEQ ID NO: 878EVQLVESGGGLVQAGGSLRLSCAASGNIFRIETMAWHRQAPGKQRELVAAIRRDEMTNYLDFVKGRFTISRDNFKNTVYLQMNSLKPEDTAVYYCNLIQRRAPYSRLETYWGQGTQVTVSS >28-G12 SEQ ID NO: 879EVQLVESGGGLVQAGGSLRLSCAASGSIFSINTMGWYRQTPGQQRDWVASISSGGTTAYADSVKGRFSISRDGPKNTVYLQMNSLKPEDTAVYYCKAQRRWSQDYWGQGTQVTVSS >28-G6 SEQ ID NO: 880EVQLVESGGGLAQAGGSLRLSCAASGSITSFNAVGWWRQAPGTQREWVASISTSGSTITPYVDSVKGRFTISRDNTKNTVYLQMNSLKPEDTAVYYCCATERMHHSYCGQGTQVTVSS >29-A12 SEQ ID NO: 881EVQLVESGGGLVQAGGSLRLSCAASGRTFSEVVMGWFRQTPGMKREFVAAISGSENVTSYADSVKGRFTISRDNAKNTVTLQMNSLKPEDTAVYYCAAQRWRGGSYEYWGQGTQVTVSS >29-A6 SEQ ID NO: 882EVQLVESGGGLVQAGGSLGLSCVFSGRPYSYDAMAWFRQAPGKEREFVAVITWSGGTTVYADSVQGRFTISRDIAKNTMNLQMNSLKPDDTAVYYCAGSAGARYGVGWWRNGQNYQNWGQGTQVTVSS >29-C6 SEQ ID NO: 883EVQLVESGGESVQVGGSVRLSCAASGLTFSNFIMGWFRRAPGKERELLAAIGDPSTHYADSATGRFTISRDNAKNMVYLQMNSLKTEDTAVYYCAARSRYSTGTLYDQTKYIYWGQGTQVTVSS >29-E12 SEQ ID NO: 884EVQLVESGGGLVQAGGSLTLSCVVSGVDFSRHVIGWFRQAPGKERKFVTAINSDGDTTTDDRSLKGRFTISRFNANNTVHLQMTNLKVEDTAIYYCATGPQYRSYFARSYLYWGQGTQVTVSS >29-F6 SEQ ID NO: 885EVQLVESGGGRVQAGGSLRLSCEASGRTFSDYIIGWFRQGPGKERESVARISGSGLTNTTTETVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAYSGHFSGRVSDFLYWGQGTQVTVSS >29-H1 SEQ ID NO: 886EVQLVESGGGLVQPGGSLRLACAASKSIDNIHAMGWYRQAPGNEREWVASITSSATAYADSVKGRFIISRDNAENTIYLQMHSLKPEDTAVYYCKGQVLVPGGRNDYWGQGTQVTVSS >41-B5, C1, D3, E4, E5 SEQ ID NO: 887EVQLVESGGGLVQAGGSLRLACAASGRTFSTYAMGWFRQAPGKEREFVAAISWNEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKREDTAVYYCASDRHYTAQQMRVMTGASYMDYWGKGTLVTVSS >30-B 12, E6 SEQ ID NO: 888EVQLVESGGGLVQAGGSLRLSCAASGPTFSIYDMGWFRQAPGKEREFVTVIRWSDGFTYYEDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYVCAANTAPLRIINFRNAYNYDYWGQGTQVTVSS >30-A6 SEQ ID NO: 889EVQLVESGGGLVQAGGSLRLSCAASGRASGDYAMGWFRQAPEKEREFVATINYSGGVTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADRHYGAYALLGLGTHAYIDYRGQGTQVTVSS >30-B1 SEQ ID NO: 890EVQLVESGGGLVPAGGSLRLSCAGSGRSFSRLAMGWFRQAPGKERDFVGAINWLSESTYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASDRFATAQNMNTMGSLDYWGQGTQVTVSS >30-C1 SEQ ID NO: 891EVQLVESGGGLVQAGDSLRLSCAVSGRTISSYAVGWFRQAPGKEREFVAVISWTGGSTYFADSVKGRFTISRDNAKNTVYLQMNNLKSEDTAVYYCTADRFTTGSGRTSYPRPSEFDHWGQGIQVTVSS >30-D1 SEQ ID NO: 892EVQLVESGGGLVQPGGSLRLSCAASGFAFSSDWMYWVRQAPGKGPEWVSSIGIAGTPTFYADSVKGRFTISRDNANNMLYLQMTSLKPGDTALYYCAREGIYCSNWRCLFGPKTDLPASWGQGTQVTVSS >30-D12 SEQ ID NO: 893EVQLVESGGGLVQAGGSLRLSCATSGRTFSDYVVGWFRQTPGKEREFIGRKVWSNGNTYYIDSVKGRFTISGDNAKRTVYLQMNSLKPEDTALYYCAARSPMSPTWDNWGQGTQVTVSS >30-E12 SEQ ID NO: 894EVQLVESGGGLVPAGGSLRLSCAGSGRTFSRLAMGWFRQAPGKEREFVAAISWLGESTYYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASDRFATAQAMGAMGSLDYWGQGTQVTVSS >30-G1 SEQ ID NO: 895EVQLVESGGGLVQAGISLKLSCTASGGTLTNYFMGWFRQAPGKEREFVAGVAWSSDFTAYTDSVKGRFTISRDNAKNTVYLQMNDVKPEDTAVWYCAARRRGGGYNQLNLYDYWGQGTQVTVSS >30-G12 SEQ ID NO: 896EVQLVESGGRLVQAGGSLRLSCAASGRTFSTDVMGWFRQVPEKEREFVAEIGWRDTTLYADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCSSRRAAAAYDQWGQGTQVTVSS >31-B1 SEQ ID NO: 897EVQLVESGGGLVQAGGSLRLACAASGRTFSTLAMGWFRQAPGKEREFVAAISWSEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKPEDTAVYYCAADRHYSAQQMRVMTGASYMDYWGKGTLVTVSS >31-D12 SEQ ID NO: 898EVQLVESGGGLVQAGDSLRLSCAPSGGIISNYHVGWFRQAPGKEREFVAAISASGTSTAYAGSVKGRFTISRDNAKNTAFLQMNTLKPEDTAVYYCAASEYVFRYYDDSRYYAYWGQGTQVTVSS >31-E12 SEQ ID NO: 899EVQLVESGGGLVQAGGSLRLSCSSSGRTYSAYNMGWFRLRPGKEREFVAAINWSGGTQDYVDSVKGRFTAIADVAKKTVFLQMTSLKPEDTAVYYCAATQWGSSGWKQARWYDFWGQGTQVTVSS >31-F6 SEQ ID NO: 900EVQLVESGGGLVQAGGSLRLACAASGRTFSTLAMGWFRQAPGKEREFVAAISWSEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKREDTAVYYCASDRHYTAQQMRVMTGASYMDYWGKGTLVTVSS >31-G12 SEQ ID NO: 901EVQLVESGGGLVQAGESLRLSCAASGRTFGNYVMGWFRQAPGKERELVAAITWSDSRTDYADSVKGRFTISRDNVKNLVYLQMNSLRPEDTAVYSCAASSIGPYRLLDSSRYAYWGRGTQVTVSS >41-A1 SEQ ID NO: 902KVQLVESGGGLVQAGGSLKLACAASGRTFSTLAMGWFRQAPGKEREFVAAISWSEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKPEDTAVYYCAADRHYSAQQMRVMTGASYMDYWGKGTLVTVSS >41-A3 SEQ ID NO: 903EVQLVESGGGLVQAGGSLRLACAASGRTFSTYAMGWFRQAPGKEREFVAAISRNEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKREDTAVYYCASDRHYTAQQMRVMTGASYMDYWGKGTLVTVSS >41-B1 SEQ ID NO: 904EVQLVESGGGLVQAGGSLRLSCTGSDRTFIGYHMGWFRQAPGKEREFVAYISSGGAYSNYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYHCAATDYNKAYAREGRRYDYWGQGTQVTVSS >41-C5 SEQ ID NO: 905EVQLVESGGGLVQAGGSLRLSCVVSGTITSAVFMGWYRQLPGKQRELVASINRGGSTNYAPSAKGRFTISRVNANSTMYLQMNSLQPEDTAVYYCYGVINTRSFWGQGTQVTVSS >41-D1 SEQ ID NO: 906EVQLVESGGGLVQAGGSLRLACAASGRTFSTLAMGWFRRAPGKEREFVAAISWSEDNTYYSDSVKGRFTISRNNAESTVYLQMNSLKPEDTAVYYCASDRHLLAQQMRVMTGASYMDYWGKGTLVTVSS >41-F6 SEQ ID NO: 907EVQLVESGGGLVQAGGSLRLACAASGRTFSTLAMGWFRQAPGKEREFVAAISWSEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKPEDTAVYYCVSDRHATAQHMRVMTGASYMDYWGKGTLVTVSS >39-A8, D7, E7, F9, G8, H8, A9SEQ ID NO: 908 EVQLVESGGGLVQTGGSLRLSCAASGRTFTSYCVGWWRQAPGKERDVVAAITRGSNSTDYVDSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAADTNCRNLYTGRPEYWGQGTQVTVSS >32-E12, G12, 39-D2, F2 SEQ ID NO: 909EVQLVESGGGLVQAGGSLRLSCAASGFTFGDYAIGWFRQAPGKEREGVSCISISDSSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADRLAYGLDPNFYDYWGQGTQVTVSS >32-E1, E6, 39-A2 SEQ ID NO: 910EVQLVESGGGLVQAGGSLRLSCAASERIFSTYFMGWFRQAPGKEREFVAFISGNGGSTDYADSVKGRFAISRDNVKNTLYLQMSSLKPDDTAVYYCAVAGRQIKSTWDYWGQGTQVTVSS >32-D1, 39-A1 SEQ ID NO: 911EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCISVSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADRLAYGLDPNFYDHWGQGTQVTVSS >39-D8, E8 SEQ ID NO: 912EVQLVESGGGLVRARGSLRLSCAASGGTFSRYAMGWFRQAPGKEREFVAAISRSGGNTYFTESVKGRFTMSRDNAKNTVYLQMNSLKPEDTAVYYCAARSAAVYTTTVYLVDFEYNYWGQGTQVTVSS >32-C6 SEQ ID NO: 913KVQLVESGGGLVQAGGSLRLSCAASGFTFGDYAIGWFRQAPGKEREGVSCISISDSSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADRLAYGLDPNFYDYWGQGTQVTVSS >32-F1 SEQ ID NO: 914EVQLVESGGGLVQAGGSLTLSCAASGRTFSSYRLGWFRQAPGNEREFVASITWSSANTYYADSVKGRFTISRERAKNTMYLQMDSLRPEDTAVYYCAKEDVGKPFDSWGQGTQVTVSS >32-G6 SEQ ID NO: 915EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCISSSDGRTYYDDSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADRLAYGLDPNFYDYWGQGTQVTVSS >33-A1 SEQ ID NO: 916EVQLVESGGGLVQAGGSLRLSCAASGDTFSRYSMGWFRQAPGKEREFVVYITRSGRTTYYQDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAMRSGNVRYPERYDYWGQGTQVTVSS >33-C6 SEQ ID NO: 917EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYYMGWFRQAPGKEREFVAHISLSGGNTEYADSVKGRFTITRDNAGNTVYLQMNSLKPEDTGVYCCAASPSLRSAWQYWGQGTQVTVSS >33-D1 SEQ ID NO: 918EVQLVESGGGLVQAGDSLRLSCAATGRTFSSDAMGWFRQAPGKERAFVAGINYNSVYRYYTDSVEGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADSDYYSLIGGRPVNYWGQGTQVTVSS >33-G6 SEQ ID NO: 919EVQLVESGGGSVQTGGSLRLSCAASGRTFTSYCVGWWRQAPGKERAVVAAITRGGDTTDYVDSVKGRFTISRDKAENTVYLQMNSLKPEDTAVYYCAADINCRNLYTGRPEYWGRGTQVTVSS >39-B1 SEQ ID NO: 920EVQLVESGGGLVQAGGSLRLSCARSGRISNINIMAWYRQAPGKTRDMVAAIIGDSTNYADSVKGRFTISRDNAKNTVYLHMNRLKPEDTGVYYCKISGVDWGQGTQVTVSS >39-B2 SEQ ID NO: 921EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCISSSDGRTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADRLAYGLDPNFYDYWGQGTQVTVSS >39-C1 SEQ ID NO: 922EVQLVESGGGLVQAGGSLRLSCSASGRTFGSYVMGWFRQAPGKEREFVAAVSRSGRNINYADLMKGRFTVSRDNTKNTVYLQLNNLTPEDTAVYYCAADGTISSSWADLRRGETYGDWGQGTQVTVSS >39-C2 SEQ ID NO: 923KVQLVESGGGLVQAGGSLRLSCAASGRTFSRYYMGWFRQAPGKEREFVAFISGTGGSIDYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCAAASGNSGRSTWDYWGQGTQVTVSS >39-E1 SEQ ID NO: 924EVQLVKSGGGLVQAGGSLRLSCAASGNIFINNAVGWYRQAPGKQREMVAAMLSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVQVNGIWARWGQGTQVTVSS >39-E2 SEQ ID NO: 925EVQLVESGGGLVQAGGSLRLSCGRSGRISNTNIMSWYRQAPGKTRDMVAAIIGDATNYADSVKGRFTISRDNAKNAVSLHMNRLKPEDTGVYYCKIPGVDWGQGTQVTVSS >39-F1 SEQ ID NO: 926EVQLVESGGGLVQAGGSLRLSCAASERIFSTYFMGWFRQAPGKEREFVAFISGNGGSTDYADSVKGRFAISRDNVKNTLYLQMSSLKPDDTAVYYCAVAGRQIKSTWGYWGQGTQVTVSS >39-G1 SEQ ID NO: 927EVQLVESGGGLVQAGGSLRLSCARSGRISNINIMSWYRQAPGKTRDMVAAIIGDSTNYADSVKGRFTISRDNAKNTVHLQMNRLKPEDTGVYYCNIPGVDWGQGTQVTVSS >39-G2 SEQ ID NO: 928EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCISMSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADRLAFGLDSNFYDYWGQGTQVTVSS >39-C8 SEQ ID NO: 929EMQLVESGGGLVQTGGSLRLSCAASGRTFTSYCVGWWRQAPGKERDVVAAITRGSNSTDYVDSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAADINCRNLYTGRPEYWGQGTQVTVSS >39-D9 SEQ ID NO: 930EVQLVESGGGLVQPGGSLRLACAASGRTFTSYCVGWWRQAPGKERDVVAAITRGSNSTDYVDSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAADINCRNLYTGRPEYWGQGTQVTVSS >39-D1 SEQ ID NO: 931KVQLVESGGGLVQAGGSLRLSCAASGNIFINNAVGWYRQAPGKQREMVAAMLSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVQVNGTWARWGQGTQVTVSS >35-B12, E1, G1, F6 SEQ ID NO: 932EVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPGKEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >35-C6, 40-E1 SEQ ID NO: 933KVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPGKEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-A2, H2 SEQ ID NO: 934EVQLVKSGGGLVQTGGSLRLSCIVSGGTFSTYAMGWFRQAPGKEREFVAGITRSGLSTSYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-B1, D1 SEQ ID NO: 935EVQLVESGGGLVQTGGSLRLSCIVSGGTFSTYAMGWFRQAPGKEREFVAGITRSGLSTSYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >35-B1, G12, 40-E2 SEQ ID NO: 936EVQLVKSGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPGKEREFVAGITRSGVSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >35-C12 SEQ ID NO: 937KVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPGKEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCATTRRLHFGNGADYWGKGTPVTVSS >35-F12 SEQ ID NO: 938EVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPGKEREFVAGITRSGVSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYSCAATRRLHFGNGADYWGKGTPVTVSS >35-G6 SEQ ID NO: 939EVQLVESGGGLVQAGDSLRLSCTASGAFGSYAIAWFRQTPGKEREFVAVINRSGSYVYYTDSVKGRFTISRHNAKNTAYLQMNSLKPEDTAVYYCAARDGTLYSTTYYYISSYTYWGQGTQVTVSS >40-A1 SEQ ID NO: 940EVQLVESGGGLVQAGGSLRLSCAASGGTFSTYAMGWFRQAPGKEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-B2 SEQ ID NO: 941EVQLVESGGGLVQTGGSLRLSCIASGGTFSTYAMGWFRQAPGKEREFVAGITRSALSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-C1 SEQ ID NO: 942EVQLVESGGGLVQAGDSLRLSCTASGTFGSYAIAWFRQTPGKEREFVAVINRSGSYVYYTDAVKGRFTISRHNAKNTAYLQMNSLKPEDTAVYYCAARDGTLYSTTYYYISSYTYWGQGTQVTVSS >40-D2 SEQ ID NO: 943EVQLVESGGGLVQPGGSLRLSCRASGGTFRKLAVAWFRQAPGKEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-F1 SEQ ID NO: 944EVQLVESRGGLVQTGGSLRLSCAASGGTFSTYAMAWFRQAPGKEREFVAGITRSAVSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-F2 SEQ ID NO: 945EVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPGKEREFVAGITRSSVSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-G1 SEQ ID NO: 946EVQLVESGGGLVQAGDSLRLSCTASGTFGSYAIAWFRQTPGKEREFVAVINRSGSYMYYIDSVKGRFTISRHNAKNTAYLQMNSLKPEDTAVYYCAARDGTLYSTSYYYISSYTYWGQGTQVTVSS >40-G2 SEQ ID NO: 947EVQQVESGGGLVQTGGSLRLSCIVSGGTFSTYAMGWFRQAPGKEREFVAGITRSGVSTSYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-H1 SEQ ID NO: 948EVQLMESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPGKEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPEDTALYYCAATRRLHFGNGADYWGKGTPVTVSS >36-A6, 40-H8 SEQ ID NO: 949EVQLVESGGGLVQAGDSLRLSCAASGRTLSSYNMGWFRQAPGKEQEFVAADMWSGTTTYYTDSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAAELHSSDYTSPGAYAYWGQGTQVTVSS >36-A1 SEQ ID NO: 950EVQLVESGGGLVQPGGSLTLSCAASGGTFSIRAMGWLRQAPGNEREFVAAISRDGDRTYYTDVVKGRFTISRDNAKSTVYLQINSLKTEDTAVYYCAATRPFKVLTATIENDFTYWGQGTQVTVSS >36-C6 SEQ ID NO: 951EVQLVESGGGLVQAGGSLRLSCAFSDGRTVSRYHMGWFRQGPGKEREFVAAVSSLGPFTRYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCAADSSGYSGSYSSEYRYDYWGQGTQVTVSS >36-D6 SEQ ID NO: 952EVQLVESGGGLVQAGGSLRLSCVASGLTFRNYAMAWFRQAPGKEREFVAGINWSGNGVYYPDSLKERFAISRENPKNMVYLQMNSLNPEDTAVYYCTADRTLTAWDRDNAEYWGQGTQVTVSS >36-E1 SEQ ID NO: 953EVQLVESGGGLVQAGDSLRLSCAASGRTLSSYNMGWFRQAPGKELEFVAAIMWSGTTTYYTDSVKGRFTISTDNAKNMVYLQMNSLKPEDTAVYYCAAELHSSDYTSPGAYAYWGQGTQVTVSS >36-F1 SEQ ID NO: 954EVQLVESGGGLVQPGGSLTLSCAASGGTFSIRAMGWFRQAPGNEREFVAAISRDGDRTYYTDVVKGRFTISRDNAKSTVYLQINSLKTEDTAVYYCAATRPFKVLSAIIENDFTYWGQGTQVTVSS >37-B1 SEQ ID NO: 955EVQLVESGGGLVQPGQSLRLSCAASGRTLNPYKVAWFRQAPGKERDFVAVIHWYGITAYADTVKGRFSISRDNAKGTVYLQMDSLKPEDTAVYYCALDSTSALGHATTDFDSWGQGTQVTVSS >37-B12 SEQ ID NO: 956EVQLVESGGGLVQAGGSLRLSCAASGRSLRNYHMAWFRQAPGKEQEFVGDFRPSGGSPYYANYADSVKGRFTIFRDNAKNTVYLQMNSLKLEDTAVYYCAADSHGGIAFMEPDEYDYWGQGTQVTVSS >37-B6 SEQ ID NO: 957EVQLVESGGGLVQAGGSLRLSCTAAGRTHSIYPIGWFRQAPGKEREFVAAIDWSGSRSYYLDSMKGRFTISGDNAKNTVYLQMNSLKPEDTGVYYCAARPVGDFADPRYRFWGQGTQVTVSS >37-C12 SEQ ID NO: 958EVQLVESRGGLVQTGGSLRLSCTTSGGTLRGYGVGWFRQGPGKDREFVAAISWSPGRTDYGDAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADSVSASYYDARNDMAYDYWGRGTQVTVSS >37-C6 SEQ ID NO: 959EVQLVESGGGVVQTGGSLRLSCTTSGGTFRNYGVGWFRQGPGRDREFVAAISWSPGRTDYGDAVKGRFTISRDNPKNTVYLQMNSLKPEDTAVYYCAADSVSASYYDARNDMAYDYWGRGTQVTVSS >37-D6 SEQ ID NO: 960EVQLVESGGGLVRAGGSLRLSCAASGRTLNPYKVAWFRQAPGKERDFVAVIHWYGITAYADTVKGRFSISRDNAQGTVKLQMDSLKPEDTAVYYCALDSTSALGHTTSDFDSWGQGTQVTVSS >37-E6 SEQ ID NO: 961EVQLVESGGGLVQAGGSLRLSCAASGRSLGTYHMAWFRQAPGKEQEFVGDLRPSGGRAGYADYADSVKGRFTIFRDNAKNTVYLQMNSLKLEDTAVYYCAADSHGGISFMEPDEYDYWGQGTQVTVSS >37-F1 SEQ ID NO: 962KVQLVESGGGLVRAGGSLRLSCAASGRTLNPYKVAWFRQAPGKERDFVAVIHWYGITAYADTVKGRFSISRDNAKGTVYLQMDSLKPEDTAVYYCALDSTSALGHATTDFDSWGQGTQVTVSS >37-F12 SEQ ID NO: 963EVQLVESGGGLVQTGGSLRLSCTTSGGTFRNYGVGWFRQGPGKDREFVAAISWSPGRTDYGDAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADSVSASYYDARNDMAYDYWGRGTQVTVSS >37-G1 SEQ ID NO: 964EVQLVESGGGLVQAGGSLRLTCAASGRTFSMGRVGWFRQDPGKEREFVAAISRSGDTTYYDDSVRDRFTTSRDNAKNTIDLRMNSLKPEDTAVYYCAAATFRAVRQDPSYSASYDYWGQGTQVTVSS >37-G6 SEQ ID NO: 965EVQLVESGGGLVRAGGSLRLSCAASGRTLNPYKVAWFRQAPGKERDFVAVIHWYGITAYADTVKGRFSISRDNAKGTVYLHMDSLKPEDTAVYYCALDSTSALGHATTDFDSWGQGTQVTVSS >40-A7 SEQ ID NO: 966EVQLVESGGGLVQAGASLRLSCGASGRTLSMYTMGWFRQAPGKERDFVAAITPINWGGRGTHIADSVKGRFTIFRDNTKNTINLQMNNLNPEDTAVYYCAAESHGSTSPRNPLQYDYWGQGTQVTVSS >40-B8 SEQ ID NO: 967EVQLVESGGGLVQAGGSLRLSCAASDSARTFTNYAIAWFRQAPGKERKFVAVVNWSTYYADSVKGRFTISRDNAKNTVNLQMNSLKPEDTAVYYCAVGDYRSDYRSPVAYNYWGQGTQVTVSS >40-D7 SEQ ID NO: 968EVQLMESGGGLVQAGGSLRLSCAASGRTISSYTMAWFRQAPGKEREFVAYVFGGGEITDYADFVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCAMECVGDVYRSRDYTYWGQGTQVTVSS >40-F7 SEQ ID NO: 969EVQLVESGGGLVQAGGSLRLSCVASGLTFRNYAMAWFRQAPGKEREFVAGINWSGNGVYYPDSLKERFAISRENAKNMVYLQMNSLNPEDTAVYYCTADRTLTAWDRDSAEYWGQGTQVTVSS >40-F8 SEQ ID NO: 970EVQLVESGGGLVQAGGSLRLSCAASGLTFRMYGSGWFRRAPGKEREFVGFINHSGGRTNYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAVPISGYINPAVYDRPGSYDYWGQGTQVTVSS >40-G7 SEQ ID NO: 971EVQLVESGGGLVQAGGSLRLSCVASGLTFRNYAMAWFRQAPGKEREFVAGINWSGNGVYYPDSLKERFAISRENSKNMVYLQMNSLNPEDTAVYYCTADRTLTAWDRESAEYWGQGTQVTVSS >40-G8 SEQ ID NO: 972EVQLVESGGGLVQAGASLRLSCGASGRTLNMYTMGWFRQAPGKERDFVAAITPINWGGRGTHIADSVKGRFTIFRDNTKNTIILQMNNLNPEDTAVYYCAAESHGSTSPRNPLQYDYWGQGTQVTVSS >40-H7 SEQ ID NO: 973EVQLVESGGGLVQAGGSLGLSCAASGRTISSYTMAWFRQAPGKEREFVAYVFGGGEITDYADFVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCAMECVGDVYRSRDYTYWGQGTQVTVSS 30-B6 SEQ ID NO: 1044EVHLVESGGGVVQAGGSLRLSCVASGLPFSEYALVWFRQAPGKEKEREFVAGFAANGINTDYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASWTPAYNDIPRHMSSMDQWGKGTLVTVSS 30-C6 SEQ ID NO: 1045EVQLVESGGGLVQAGGSLRLSCAASGSIFSSNTMAWYRQAPRKQREFVASIMTDGTITYADSVKGRFAISRDTAKNTVALQMNSLKPEDTAVYYCNARQYGEYWQAAGSWGQGTQVTVSS x30-D6 SEQ ID NO: 1046EVqLVESGGGLVQPGGSLRLSCAGSGRTFSRLAMGWFRQAPGKEREFVAAISWLAETTYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASDRFATAQHMGAMGSLDYWGQGTQVTVSS 30-F12 SEQ ID NO: 1047EVQLVESGGGLVQPGGSLRLSCAASGFTLDSYAIAWFRQAPGKEREGVSCISSSDGTTYYADSVKGRFTISRDNAKNMVYLQMQSLKPEDTAVYYCAADPELVTVCRPGWGPAYDYWGQGTQVTVSS 34-A12 SEQ ID NO: 1048EVQLVESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGIRYYIDSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAADPIRICSSQPRRYDYWGQGTQVTVSS 34-H1 SEQ ID NO: 1049EVQLVESGGGLVQDGGSLSCAASGRTFSSYVMGWFRQAPGKEREFVATISRSGESTYYTGSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAADYNPYQSGSYYSSRSSTYDYWGQGTQVTVSS 35-A12 SEQ ID NO: 1050EVQLVESGGGLVQAGGSLRLSCVASGLSFDDYAIGWFRQAPGKEREGVSCIGDKGDRIFYADSVKGRFAISSDHAKHTVDLQMTNLKPEDTATYYCAAVAPFAFCTESLPDTWYDYWGQGIQVTVSS 35-D1 SEQ ID NO: 1051EVQLVESGGGLVQAGGSLRLSCAASGRTFSTYAMAWFRRAPGKEREFVVAIRWSGDRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCALRRFSGFDYSGNYYAWDAYDYWGQGTQVTVSS 35-H1 SEQ ID NO: 1052EVQLVESGGGWVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAGINGSGNGIRIALSVRGRFTITRDNAKNTGYLQMNSLKREDTAVYYCGPTFAAGASEYHYWGQGTQVTVSS x40-E8 SEQ ID NO: 1053EVQLVESGGTLVXAGGSLRLSCAASGLAFNAYGTGWFRQAPGKEREFVATISWSGSTTSYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAIYYCAMNRYGLVYKEERHYDFWGQGTQVTVSS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety for the purpose and information indicated in the specification.

1. Amino acid sequence that is directed against and/or that canspecifically bind to a proteinase from the ADAM family. 2.-3. (canceled)4. Amino acid sequence according to claim 1, that can specifically bindto a proteinase from the ADAM family with a dissociation constant(K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to10⁻¹² moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter.5. Amino acid sequence according to claim 1, that can specifically bindto a proteinase from the ADAM family with a rate of association(k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferablybetween 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.
 6. Amino acidsequence according to claim 1, that can specifically bind to aproteinase from the ADAM family with a rate of dissociation (k_(off)rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹, preferably between 10⁻² s⁻¹ and 10⁻⁶s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴s⁻¹ and 10⁻⁶ s⁻¹.
 7. Amino acid sequence according to claim 1, that canspecifically bind to a proteinase from the ADAM family with an affinityless than 500 nM, preferably less than 200 nM, more preferably less than10 nM, such as less than 500 pM.
 8. (canceled)
 9. Amino acid sequenceaccording to claim 1, that comprises an immunoglobulin fold or thatunder suitable conditions is capable of forming an immunoglobulin fold.10.-13. (canceled)
 14. Amino acid sequence according to claim 1, thatessentially consists of a light chain variable domain sequence (e.g. aVL-sequence); or of a heavy chain variable domain sequence (e.g. aVH-sequence).
 15. Amino acid sequence according to claim 1, thatessentially consists of a heavy chain variable domain sequence that isderived from a conventional four-chain antibody or that essentiallyconsist of a heavy chain variable domain sequence that is derived fromheavy chain antibody.
 16. Amino acid sequence according to claim 1, thatessentially consists of a domain antibody (or an amino acid sequencethat is suitable for use as a domain antibody), of a single domainantibody (or an amino acid sequence that is suitable for use as a singledomain antibody), of a “dAb” (or an amino acid sequence that is suitablefor use as a dAb) or of a Nanobody™ (including but not limited to a VHHsequence).
 17. (canceled)
 18. Amino acid sequence according to claim 1,that essentially consists of a Nanobody® that i) has 80% amino acididentity with at least one of the amino acid sequences of SEQ ID NO's: 1to 22, in which for the purposes of determining the degree of amino acididentity, the amino acid residues that form the CDR sequences aredisregarded; and in which: ii) preferably one or more of the amino acidresidues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108according to the Kabat numbering are chosen from the Hallmark residuesmentioned in Table A-3.
 19. Amino acid sequence according to claim 1,that essentially consists of a Nanobody® that i) has 80% amino acididentity with at least one of the amino acid sequences of SEQ ID NO's:868 to 973 and/or 1044 to 1053, in which for the purposes of determiningthe degree of amino acid identity, the amino acid residues that form theCDR sequences are disregarded; and in which: ii) preferably one or moreof the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103,104 and 108 according to the Kabat numbering are chosen from theHallmark residues mentioned in Table A-3.
 20. (canceled)
 21. Amino acidsequence according to claim 1, that in addition to the at least onebinding site for binding against a proteinase from the ADAM family,contain one or more further binding sites for binding against otherantigens, proteins or targets.
 22. Compound or construct, that comprisesor essentially consists of one or more amino acid sequences according toclaim 1, and optionally further comprises one or more other groups,residues, moieties or binding units, optionally linked via one or morelinkers.
 23. Compound or construct according to claim 22, in which saidone or more other groups, residues, moieties or binding units are aminoacid sequences. 24.-25. (canceled)
 26. Compound or construct accordingto claim 23, in which said one or more other groups, residues, moietiesor binding units are chosen from the group consisting of domainantibodies, amino acid sequences that are suitable for use as a domainantibody, single domain antibodies, amino acid sequences that aresuitable for use as a single domain antibody, “dAb”'s, amino acidsequences that are suitable for use as a dAb, or Nanobodies. 27.(canceled)
 28. Compound or construct according to claim 22, in whichsaid one or more amino acid sequences of the invention are chosen fromthe group consisting of domain antibodies, amino acid sequences that aresuitable for use as a domain antibody, single domain antibodies, aminoacid sequences that are suitable for use as a single domain antibody,“dAb”'s, amino acid sequences that are suitable for use as a dAb, orNanobodies.
 29. (canceled)
 30. Compound or construct according to claim22, which is a multivalent construct.
 31. Compound or constructaccording to claim 22, which is a multispecific construct.
 32. Compoundor construct according to claim 22, which has an increased half-life,compared to the corresponding amino acid sequence.
 33. Compound orconstruct according to claim 32, in which said one or more other groups,residues, moieties or binding units provide the compound or constructwith increased half-life, compared to the corresponding amino acidsequence.
 34. Compound or construct according to claim 33, in which saidone or more other groups, residues, moieties or binding units thatprovide the compound or construct with increased half-life is chosenfrom the group consisting of serum proteins or fragments thereof,binding units that can bind to serum proteins, an Fc portion, and smallproteins or peptides that can bind to serum proteins.
 35. Compound orconstruct according to claim 33, in which said one or more other groups,residues, moieties or binding units that provide the compound orconstruct with increased half-life is chosen from the group consistingof human serum albumin or fragments thereof.
 36. Compound or constructaccording to claim 34, in which said one or more other groups, residues,moieties or binding units that provides the compound or construct withincreased half-life are chosen from the group consisting of bindingunits that can bind to serum albumin (such as human serum albumin) or aserum immunoglobulin (such as IgG).
 37. Compound or construct accordingto claim 36, in which said one or more other groups, residues, moietiesor binding units that provides the compound or construct with increasedhalf-life are chosen from the group consisting of domain antibodies,amino acid sequences that are suitable for use as a domain antibody,single domain antibodies, amino acid sequences that are suitable for useas a single domain antibody, “dAb”'s, amino acid sequences that aresuitable for use as a dAb, or Nanobodies that can bind to serum albumin(such as human serum albumin) or a serum immunoglobulin (such as IgG).38. Compound or construct according to claim 37, in which said one ormore other groups, residues, moieties or binding units that provides thecompound or construct with increased half-life is a Nanobody that canbind to serum albumin (such as human serum albumin) or a serumimmunoglobulin (such as IgG).
 39. Compound or construct according toclaim 32, that has a serum half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence.
 40. Compound or construct accordingto claim 32, that has a serum half-life that is increased with more than1 hours, preferably more than 2 hours, more preferably more than 6hours, such as more than 12 hours, or even more than 24, 48 or 72 hours,compared to the corresponding amino acid sequence.
 41. Compound orconstruct according to claim 32, that has a serum half-life in human ofat least about 12 hours, preferably at least 24 hours, more preferablyat least 48 hours, even more preferably at least 72 hours or more; forexample, of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).
 42. Monovalent construct, comprising oressentially consisting of one amino acid sequence according to claim 1.43. Monovalent construct according to claim 42, in which said amino acidsequence of the invention is chosen from the group consisting of domainantibodies, amino acid sequences that are suitable for use as a domainantibody, single domain antibodies, amino acid sequences that aresuitable for use as a single domain antibody, “dAb”'s, amino acidsequences that are suitable for use as a dAb, or Nanobodies. 44.(canceled)
 45. Nucleic acid or nucleotide sequence, that encodes anamino acid sequence according to claim
 1. 46.-47. (canceled)
 48. Methodfor producing an amino acid sequence, said method comprising the stepsof: a) expressing, in a suitable host cell or host organism or inanother suitable expression system, a nucleic acid or nucleotidesequence according to claim 45 optionally followed by: b) isolatingand/or purifying the amino acid sequence.
 49. (canceled) 50.Composition, comprising at least one amino acid sequence according toclaim
 1. 51.-52. (canceled)
 53. Method for the prevention and/ortreatment of at least one ADAM-related disease or disorder, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of at least one amino acid sequenceaccording to claim
 1. 54. Method for the prevention and/or treatment ofat least one disease or disorder that is associated with a proteinasefrom the ADAM family, with its biological or pharmacological activity,and/or with the biological pathways or signaling in which a proteinasefrom the ADAM family is involved, said method comprising administering,to a subject in need thereof, a pharmaceutically active amount of atleast one amino acid sequence according to claim
 1. 55. (canceled) 56.Method for immunotherapy, said method comprising administering, to asubject in need thereof, a pharmaceutically active amount of at leastone amino acid sequence according to claim
 1. 57. (canceled)